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004db1df-dc76-42af-952f-1f5e82b7822b.116	2.1. Characterizations of Bare CeO<sup>2</sup> and Grey (Modified) CeO<sup>2</sup> The first difference between the bare CeO<sup>2</sup> and the CeO<sup>2</sup> exposed to the solar irradiation (for 3 h) in H<sup>2</sup> flow at room temperature, is the change of the powder color. Figure 1 reports two photos of the synthetized materials: un-modified CeO<sup>2</sup> appears yellow in color, while modified CeO<sup>2</sup> is grey. This latter sample is coded as "grey CeO2". Interestingly, we have noted that only the contemporaneous treatment with solar irradiation and H<sup>2</sup> flow could obtain the grey CeO2, whereas each single treatment alone did not alter the structural and chemical properties of bare CeO2. 2.1. Characterizations of Bare CeO<sup>2</sup> and Grey (Modified) CeO<sup>2</sup> The first difference between the bare CeO<sup>2</sup> and the CeO<sup>2</sup> exposed to the solar irradiation (for 3 h) in H<sup>2</sup> flow at room temperature, is the change of the powder color. Figure 1 reports two photos of the synthetized materials: un-modified CeO<sup>2</sup> appears yellow in color, while modified CeO<sup>2</sup> is grey. This latter sample is coded as "grey CeO2". Interestingly, we have noted that only the contemporaneous treatment with solar irradiation and H<sup>2</sup> flow could obtain the grey CeO2, whereas each single treatment alone did not alter the structural and chemical properties of bare CeO2. Figure 1. Photo of the as-synthetized powders. Figure 1. Photo of the as-synthetized powders. The differences in the physico-chemical properties of bare CeO<sup>2</sup> and grey CeO<sup>2</sup> were illustrated in Figure 2, where the XRD patterns (Figure 2a), the Raman (Figure 2b), and the FTIR (Figures 2c-2d) spectra are reported. The differences in the physico-chemical properties of bare CeO<sup>2</sup> and grey CeO<sup>2</sup> were illustrated in Figure 2, where the XRD patterns (Figure 2a), the Raman (Figure 2b), and the FTIR (Figure 2c,d) spectra are reported. Both the samples exhibited the typical XRD pattern of ceria in the fluorite crystalline phase (Figure 2a), with the reflections at 2θ values of 28.6 (1 1 1), 33. 1 (2 0 0), 47.4 (2 2 0), and 56.4 (3 1 1) [34]. No substantial variation was detected in the grey CeO<sup>2</sup> compared to bare oxide, apart from a slight intensity decrease and a difference in the average crystallite size: 6.8 ± 0.8 nm for bare CeO<sup>2</sup> respect to 11.3 ± 1.1 nm for grey CeO2, calculated using the Scherrer equation on the main diffraction formation of CeO2-xdefects. bands at 1071 and 839 cm-1 CeO<sup>2</sup> was blue-shifted by 5 cm-1 Specifically, for bare CeO2, the high intense band at 1385 cm-1 can be reasonably related to the more defective surface of grey CeO2. observed in the grey CeO<sup>2</sup> can be reasonable connected to a decrease in crystallinity due to the vibration of the cubic fluorite structure [36]. The position of this peak is influenced by the distortion of the Ce-O bonds [32]. Consequently, the treatment of grey CeO<sup>2</sup> led to a more defective structure with the modification of the cubic structure of CeO2, resulting in the Raman shift. However, in the as-synthesized bare CeO2, an imperfect crystalline stoichiometry was detected, being the small shoulder at about 600 cm-1 (more intense in the bare CeO2), ascribed to Frenkel-type oxygen vacancies [37]. Other differences can be seen in the FTIR spectra (Figure 2c). The bands at about and 1620 cm-1 are attributed to the stretching and the bending of the O-H groups of residual water molecules respectively, whereas the group of bands in the range 1600-500 cm-1 are related to the presence of carbonates due to the interaction of the atmospheric carbon dioxide with ceria [38]. From the zoomed spectra illustrated in Figure 2d, it is possible to note the formation of different carbonate species. whereas the bands at 1190 and 1120 cm-1 are related to the bridged carbonate species. Finally, the intense bands at 1012 and 872 cm-1 can be also be assigned for this sample to the presence of hydrocarbonates, whereas there is no evidence of the formation of monodentate carbonates. It is clear band assigned to the monodentate carbonate was broader and shifted at 1395 cm-1 indicated the formation of hydrocarbonates [39–41]. In the grey CeO2, the Interestingly, analyzing the Raman spectra of the samples (Figure 2b), the peak at 461 cm-1 of the in the grey CeO2. The Raman peak at 461 cm-1 identifies the F2g skeletal is due to monodentate carbonates, , whereas the low Figure 2. (a) XRD patterns, (b) Raman spectra, (c) FTIR spectra of the synthetized samples, and (d) FTIR zoom of the "carbonate" zone. Both the samples exhibited the typical XRD pattern of ceria in the fluorite crystalline phase (Figure 2a), with the reflections at 2θ values of 28.6 (1 1 1), 33. 1 (2 0 0), 47.4 (2 2 0), and 56.4 (3 1 1) [34]. No substantial variation was detected in the grey CeO<sup>2</sup> compared to bare oxide, apart from a slight intensity decrease and a difference in the average crystallite size: 6.8 ± 0.8 nm for bare CeO<sup>2</sup> respect to 11.3 ± 1.1 nm for grey CeO2, calculated using the Scherrer equation on the main diffraction peak of ceria 2θ = 28.6 (1 1 1). This size enhancement of grey CeO<sup>2</sup> was related to the occurrence of the formation of defects inside the crystalline structure of CeO<sup>2</sup> caused by the solar irradiation in the H<sup>2</sup> stream. Furthermore, in accordance with the literature data [26,35], the intensity diminution observed in the grey CeO<sup>2</sup> can be reasonable connected to a decrease in crystallinity due to the formation of CeO2-x defects. Interestingly, analyzing the Raman spectra of the samples (Figure 2b), the peak at 461 cm−<sup>1</sup> of the CeO<sup>2</sup> was blue-shifted by 5 cm−<sup>1</sup> in the grey CeO2. The Raman peak at 461 cm−<sup>1</sup> identifies the F2g skeletal vibration of the cubic fluorite structure [36]. The position of this peak is influenced by the distortion of the Ce-O bonds [32]. Consequently, the treatment of grey CeO<sup>2</sup> led to a more defective structure with the modification of the cubic structure of CeO2, resulting in the Raman shift. However, in the as-synthesized bare CeO2, an imperfect crystalline stoichiometry was detected, being the small shoulder at about 600 cm−<sup>1</sup> (more intense in the bare CeO2), ascribed to Frenkel-type oxygen vacancies [37]. Other differences can be seen in the FTIR spectra (Figure 2c). The bands at about and 1620 cm−<sup>1</sup> are attributed to the stretching and the bending of the O-H groups of residual water molecules respectively, whereas the group of bands in the range 1600–500 cm−<sup>1</sup> are related to the presence of carbonates due to the interaction of the atmospheric carbon dioxide with ceria [38]. From the zoomed spectra illustrated in Figure 2d, it is possible to note the formation of different carbonate species. Specifically, for bare CeO2, the high intense band at 1385 cm−<sup>1</sup> is due to monodentate carbonates, whereas the bands at 1190 and 1120 cm−<sup>1</sup> are related to the bridged carbonate species. Finally, the bands at 1071 and 839 cm−<sup>1</sup> indicated the formation of hydrocarbonates [39–41]. In the grey CeO2, the band assigned to the monodentate carbonate was broader and shifted at 1395 cm−<sup>1</sup> , whereas the low intense bands at 1012 and 872 cm−<sup>1</sup> can be also be assigned for this sample to the presence of hydrocarbonates, whereas there is no evidence of the formation of monodentate carbonates. It is clear that the surface interaction sites in the grey CeO<sup>2</sup> were changed compared to un-treated CeO2. This can be reasonably related to the more defective surface of grey CeO2. Figure 2. (a) XRD patterns, (b) Raman spectra, (c) FTIR spectra of the synthetized samples, and (d) FTIR zoom of the "carbonate" zone. The textural properties of the CeO<sup>2</sup> samples are displayed in the Figure 3. Both the materials displayed a N<sup>2</sup> adsorption–desorption isotherm of type III, with a H3 hysteresis loop (Figure 3a), indicating the presence of macro-meso slit-shaped pores [42]. The treatment with solar lamp in H<sup>2</sup> flow led to a decrease in the Brunauer–Emmett–Teller (BET) surface area. The grey CeO<sup>2</sup> exhibited a lower surface area (67 ± 1 m2/g) than CeO<sup>2</sup> (81 ± 1 m2/g). This decrease can be reasonably due to the agglomeration of CeO<sup>2</sup> particles caused by the irradiation treatment under solar lamp in H<sup>2</sup> flow, as Catalysts 2020, 10, x FOR PEER REVIEW 5 of 16 The textural properties of the CeO<sup>2</sup> samples are displayed in the Figure 3. Both the materials displayed a N<sup>2</sup> adsorption–desorption isotherm of type III, with a H3 hysteresis loop (Figure 3a), indicating the presence of macro-meso slit-shaped pores [42]. The treatment with solar lamp in H<sup>2</sup> flow led to a decrease in the Brunauer–Emmett–Teller (BET) surface area. The grey CeO<sup>2</sup> exhibited a lower surface area (67 ± 1 m<sup>2</sup> /g) than CeO<sup>2</sup> (81 ± 1 m<sup>2</sup> /g). This decrease can be reasonably due to the agglomeration of CeO<sup>2</sup> particles caused by the irradiation treatment under solar lamp in H<sup>2</sup> flow, as further confirmed by the increase in mean crystalline size calculated by XRD. As a consequence, it was verified a shift towards large pores in the Barrett, Joyner and Halenda (BJH) pore size distribution curves (Figure 3b), with the mean pore size of the grey CeO<sup>2</sup> higher (58 ± 1 nm) with respect to bare CeO<sup>2</sup> (36 ± 1 nm). This size increase, verified by the grey CeO2, is strictly correlated with the peculiar treatment of this latter sample, i.e., the simultaneous utilization of the simulated solar radiation and the H<sup>2</sup> stream. As stated before, according to the work of Aslam et al. [32], the solar light alone did not caused any change in the mean size of bare CeO2; however, we used a more focused solar lamp than in ref. [32], which led to a slight heating of the sample (from room temperature to about 40 ◦C). On the contrary, the irradiation in a reductive atmosphere (H<sup>2</sup> stream) promoted the formation of numerous oxygen vacancies, a process characterized with an increase in the internal pressure inside the ceria crystalline structure with a consequent interatomic bond cleavage [43]. Reasonably, this process resulted in an agglomeration with a measurable size increase in the grey CeO<sup>2</sup> particle size. The same linear correlation between the increase in mean crystalline size, and the decrease in the BET surface area was already reported in the literature with other CeO2-based samples [44,45]. The formation of defects did not alter the morphology of the CeO<sup>2</sup> materials, that, if prepared by chemical precipitation, are usually characterized by a random stacking of particles [23,32]. further confirmed by the increase in mean crystalline size calculated by XRD. As a consequence, it was verified a shift towards large pores in the Barrett, Joyner and Halenda (BJH) pore size distribution curves (Figure 3b), with the mean pore size of the grey CeO<sup>2</sup> higher (58 ± 1 nm) with respect to bare CeO<sup>2</sup> (36 ± 1 nm). This size increase, verified by the grey CeO2, is strictly correlated with the peculiar treatment of this latter sample, i.e., the simultaneous utilization of the simulated solar radiation and the H<sup>2</sup> stream. As stated before, according to the work of Aslam et al. [32], the solar light alone did not caused any change in the mean size of bare CeO2; however, we used a more focused solar lamp than in ref. [32], which led to a slight heating of the sample (from room temperature to about 40 °C). On the contrary, the irradiation in a reductive atmosphere (H<sup>2</sup> stream) promoted the formation of numerous oxygen vacancies, a process characterized with an increase in the internal pressure inside the ceria crystalline structure with a consequent interatomic bond cleavage [43]. Reasonably, this process resulted in an agglomeration with a measurable size increase in the grey CeO2 particle size. The same linear correlation between the increase in mean crystalline size, and the decrease in the BET surface area was already reported in the literature with other CeO2 based samples [44,45]. The formation of defects did not alter the morphology of the CeO<sup>2</sup> materials, that, if prepared by chemical precipitation, are usually characterized by a random stacking of particles [23,32]. The UV-vis Diffuse Reflectance spectra of the CeO<sup>2</sup> powders are displayed in Figure 4a where the reflectance function (Kubelka–Munk function) is plotted versus the wavelength. A slight variation in the absorption features was detected for grey CeO<sup>2</sup> with a shift at lower wavelengths that results in a slightly higher optical band-gap (3.1 ± 0.3 eV) compared to bare CeO<sup>2</sup> (2.7 ± 0.3 eV) estimated by graphing the modified Kubelka–Munk function versus the eV (Figure 4b) [46]. The lower band-gap of CeO<sup>2</sup> (activation wavelength ≤ 460 nm) is suitable to exploit, together with the UV portion, a part of visible component of the solar light, whereas the grey CeO<sup>2</sup> with a higher band-gap (activation wavelength ≤ 400 nm) will be preferentially activated by the solar UV photons. Figure 3. (a) N<sup>2</sup> adsorption–desorption isotherms of the CeO<sup>2</sup> samples; (b) pore size distribution curve of the analyzed samples evaluated by means of the Barrett, Joyner and Halenda (BJH) method. Figure 3. (a) N<sup>2</sup> adsorption–desorption isotherms of the CeO<sup>2</sup> samples; (b) pore size distribution curve of the analyzed samples evaluated by means of the Barrett, Joyner and Halenda (BJH) method. The UV-vis Diffuse Reflectance spectra of the CeO<sup>2</sup> powders are displayed in Figure 4a where the reflectance function (Kubelka–Munk function) is plotted versus the wavelength. A slight variation in the absorption features was detected for grey CeO<sup>2</sup> with a shift at lower wavelengths that results in a slightly higher optical band-gap (3.1 ± 0.3 eV) compared to bare CeO<sup>2</sup> (2.7 ± 0.3 eV) estimated by graphing the modified Kubelka–Munk function versus the eV (Figure 4b) [46]. The lower band-gap of CeO<sup>2</sup> (activation wavelength ≤ 460 nm) is suitable to exploit, together with the UV portion, a part of visible component of the solar light, whereas the grey CeO<sup>2</sup> with a higher band-gap (activation wavelength ≤ 400 nm) will be preferentially activated by the solar UV photons. Catalysts 2020, 10, x FOR PEER REVIEW 6 of 16 Figure 4. (a) UV-vis Diffuse Reflectance spectra of CeO<sup>2</sup> powders; (b) estimation of the optical bandgap of the samples by means of the modified Kubelka–Munk function. Figure 4. (a) UV-vis Diffuse Reflectance spectra of CeO<sup>2</sup> powders; (b) estimation of the optical band-gap of the samples by means of the modified Kubelka–Munk function. For the photocatalytic degradation of the IMI and especially for the Fenton and photo-Fenton reactions, it is fundamental that the presence of Ce3+ defects on the surface of CeO2. To establish the presence of these defect states, the XPS analysis was performed and the results are illustrated in Figure 5. In accordance with the literature data, the Ce 3d5/2 state involves the v, v', v'' and v''' component, whereas the u, u', u'' and u''' components are related to the Ce 3d3/2 state [47–50]. The v' and u' components indicate the presence of Ce3+ , whereas the peak at 916.4 eV (u''') for CeO<sup>2</sup> and at 916.8 eV for the grey CeO<sup>2</sup> are the typical fingerprint of Ce4+ [48–50]. It is clearly visible from Figure 5, as the component v' at 885.2 eV of Ce3+ is intense for grey CeO<sup>2</sup> whereas the same signal is absent in the bare CeO2. The u' signal is covered to the u and u'' components in both the samples. Furthermore, it is possible to note that, as the ratio between the v''' and u components is different and shifted of about 0.5 eV, as for the v component, compared to un-modified CeO2. This is another indication of the modification of the ceria surface sites with the higher presence of Ce3+ states in the grey CeO<sup>2</sup> [50]. The irradiation with solar lamp in H<sup>2</sup> stream thus induced the formation of CeO2-x defects on the surface of CeO2, as also confirmed by the Raman spectroscopy, with a consequent modification to the surface chemical composition of ceria, as also indirectly corroborated by the FTIR with the formation of different carbonate species in the two CeO<sup>2</sup> samples. For the photocatalytic degradation of the IMI and especially for the Fenton and photo-Fenton reactions, it is fundamental that the presence of Ce3<sup>+</sup> defects on the surface of CeO2. To establish the presence of these defect states, the XPS analysis was performed and the results are illustrated in Figure 5. In accordance with the literature data, the Ce 3d5/<sup>2</sup> state involves the v, v', v" and v"' component, whereas the u, u', u" and u"' components are related to the Ce 3d3/<sup>2</sup> state [47–50]. The v' and u' components indicate the presence of Ce3+, whereas the peak at 916.4 eV (u"') for CeO<sup>2</sup> and at 916.8 eV for the grey CeO<sup>2</sup> are the typical fingerprint of Ce4<sup>+</sup> [48–50]. It is clearly visible from Figure 5, as the component v' at 885.2 eV of Ce3<sup>+</sup> is intense for grey CeO<sup>2</sup> whereas the same signal is absent in the bare CeO2. The u' signal is covered to the u and u" components in both the samples. Furthermore, it is possible to note that, as the ratio between the v"' and u components is different and shifted of about 0.5 eV, as for the v component, compared to un-modified CeO2. This is another indication of the modification of the ceria surface sites with the higher presence of Ce3<sup>+</sup> states in the grey CeO<sup>2</sup> [50]. The irradiation with solar lamp in H<sup>2</sup> stream thus induced the formation of CeO2-x defects on the surface of CeO2, as also confirmed by the Raman spectroscopy, with a consequent modification to the surface chemical composition of ceria, as also indirectly corroborated by the FTIR with the formation of different carbonate species in the two CeO<sup>2</sup> samples. (a) (b) Figure 4. (a) UV-vis Diffuse Reflectance spectra of CeO<sup>2</sup> powders; (b) estimation of the optical bandgap of the samples by means of the modified Kubelka–Munk function. For the photocatalytic degradation of the IMI and especially for the Fenton and photo-Fenton reactions, it is fundamental that the presence of Ce3+ defects on the surface of CeO2. To establish the presence of these defect states, the XPS analysis was performed and the results are illustrated in Figure 5. In accordance with the literature data, the Ce 3d5/2 state involves the v, v', v'' and v''' component, whereas the u, u', u'' and u''' components are related to the Ce 3d3/2 state [47–50]. The v' and u' components indicate the presence of Ce3+ , whereas the peak at 916.4 eV (u''') for CeO<sup>2</sup> and at 916.8 eV for the grey CeO<sup>2</sup> are the typical fingerprint of Ce4+ [48–50]. It is clearly visible from Figure 5, as the component v' at 885.2 eV of Ce3+ is intense for grey CeO<sup>2</sup> whereas the same signal is absent in the bare CeO2. The u' signal is covered to the u and u'' components in both the samples. Furthermore, it is possible to note that, as the ratio between the v''' and u components is different and shifted of about 0.5 eV, as for the v component, compared to un-modified CeO2. This is another indication of the modification of the ceria surface sites with the higher presence of Ce3+ states in the grey CeO<sup>2</sup> [50]. The irradiation with solar lamp in H<sup>2</sup> stream thus induced the formation of CeO2-x defects on the surface of CeO2, as also confirmed by the Raman spectroscopy, with a consequent modification to the surface chemical composition of ceria, as also indirectly corroborated by the FTIR with the formation of different carbonate species in the two CeO<sup>2</sup> samples. the IMI pesticide. Three different AOPs were investigated: a) the photocatalytic oxidation (Figure 6a) Figure 5. XPS spectra of the CeO<sup>2</sup> samples. Figure 5. XPS spectra of the CeO<sup>2</sup> samples. We have compared the (photo)catalytic activity of the synthetized sample in the degradation of 2.2. (Photo)catalytic Activity 2.2. (Photo)catalytic Activity ### 2.2. (Photo)catalytic Activity way, crucial steps. Interestingly, in the photo-Fenton like reaction (Figure 6c), the grey CeO<sup>2</sup> displayed the best We have compared the (photo)catalytic activity of the synthetized sample in the degradation of the IMI pesticide. Three different AOPs were investigated: a) the photocatalytic oxidation (Figure 6a) utilized as an irradiation source a solar lamp; b) the Fenton reaction (Figure 6b), adding 5 mL of H2O<sup>2</sup> (3%, 0.9M) in the reaction mixture, c) the photo-Fenton reaction (Figure 6c) utilizing both the solar lamp and the hydrogen peroxide. performance (~35% of degradation), comparing all the investigated AOPs with the two CeO<sup>2</sup> samples. The solar irradiation could boost the further formation of Ce3+ sites. An indirect confirmation is derived from the slight enhancement of the catalytic activity of bare CeO<sup>2</sup> (25%) compared to the solar photocatalytic test (20%) that can be attributed to the formation of in situ oxygen vacancies in the surface of bare CeO<sup>2</sup> which can react with the hydrogen peroxide. Catalysts 2020, 10, x FOR PEER REVIEW 8 of 16 these tests, Figure S1). The regeneration and the further formation of Ce3+ defect centres are, in this Figure 6. (a) Photocatalytic degradation of imidacloprid (IMI) under solar light irradiation, (b) Fenton-like reaction, (c) photo-Fenton like reaction on the CeO2-based samples, (d) photo-Fenton like reaction utilizing grey CeO<sup>2</sup> in different runs. Figure 6. (a) Photocatalytic degradation of imidacloprid (IMI) under solar light irradiation, (b) Fenton-like reaction, (c) photo-Fenton like reaction on the CeO<sup>2</sup> -based samples, (d) photo-Fenton like reaction utilizing grey CeO<sup>2</sup> in different runs. As reported in the literature [25,32], the irradiation of ceria with photons which possess energy higher than the CeO2 band-gap can exploit the following reaction: CeO<sup>2</sup> + hν (E ≥ Eg) → Ce+3,+4O2-x + x/2 O<sup>2</sup> (9) The interaction of the highly energetic photons with the surface of CeO<sup>2</sup> leads to the loss of surface oxygen, thus allowing the formation of the Ce3+ states. The same reaction was exploited during the preparation of grey CeO2, where the formation of Ce3+ was further increased due to the reducing atmosphere. Therefore, with the grey CeO2, owing to a higher number of defective centres compared to bare ceria (as shown by XPS and Raman analyses), it is possible to reach the best performance in the degradation of IMI by the photo-Fenton-like reaction. Furthermore, the The solar photodegradation of pesticides required harder conditions in comparison to the degradation of other pollutants (for example, dyes) [51,52]. As a result, even utilizing TiO<sup>2</sup> (the most investigated photocatalyst), the degradation efficiency is not so high [7,53]. Furthermore, in accordance with our preceding work [7], and the literature data [54,55], as confirmed for all the AOPs investigated, the IMI degradation is characterized by the formation of various by-products as amine and chloro-pyridine species. The reaction mechanism involves the breaking of C–N and the N–N bonds followed by the formation of small molecules, such as chlorine dioxide, nitrogen oxides species, water and carbon dioxide [7,54,55]. The reported degradation percentage of IMI (i.e., the variation of the IMI concentration respect to the initial IMI concentration) was low even through photocatalysis [54–56], solar photo-Fenton [57], or UV-A photolysis [28]. In particular, with UV irradiation it is possible to obtain a complete photolysis of IMI after contemporaneous presence of the hydrogen peroxide and the solar irradiation enhances the a long time of irradiation (about 10 h) [28], whereas in our precedent work [7] with molecularly imprinted TiO<sup>2</sup> samples, it was possible to selectively photodegrade IMI even in a pesticide mixture, although the degradation efficiency did not exceed 40% with a partial mineralization of ~35% (evaluated by the Total Organic Carbon, TOC, analysis) after 3 h of UV irradiation. Kitsiou et al. [57] found that the reaction efficiency can be improved utilizing a combination of photo-Fenton under UV-A irradiation and TiO2, due to the synergism between the homogenous iron catalyst and the heterogeneous TiO<sup>2</sup> photocatalyst (~80% of degradation after 2 h of UV-A irradiation and ~60% of TOC mineralization), whereas only the solar homogenous photo-Fenton with iron reached ~50% for both degradation and removal of organic carbon after 3 h of UV-A irradiation. The most promising result was obtained by Sharma et al. [54] with a particular TiO<sup>2</sup> supported on mesoporous silica SBA-15, that allowed to achieve ~90% IMI degradation after 3 h of solar irradiation. In this contest, the obtained (photo)catalytic performances of CeO<sup>2</sup> for the degradation of IMI described in this work are in line with the results obtained with the TiO2-based materials. Figure 6a reports the photocatalytic degradation of the synthetized powders. In the test without catalysts, (black line in Figure 6a) no substantial variations in the initial concentration of IMI was measured, as expected. On the other hand, after 3 h of solar light irradiation the bare CeO<sup>2</sup> was able to degrade around the 20% of the initial concentration of IMI, whereas the grey CeO<sup>2</sup> showed a slightly lower performance (~16%). This can be reasonably explained considering the lower surface area and/or the slightly higher band-gap of grey CeO<sup>2</sup> with respect to the un-modified CeO2. The catalytic activity through the Fenton reaction (Figure 6b) is significantly lower compared to the photocatalytic tests (the test was carried out without irradiation). In these tests, no substantial degradation of IMI was measured in the run carried out without catalysts, but with H2O<sup>2</sup> (Figure 6b, olive line). As explained in the Introduction (see reactions 6-8), the Fenton process requires the presence and the fast regeneration of Ce3<sup>+</sup> defect sites. For this reason, differently to the photocatalytic tests, the grey CeO<sup>2</sup> is more active than the bare CeO2. As detected by Raman and XPS measurements, the un-modified CeO<sup>2</sup> exhibited a much lower presence of defect centres with respect to grey CeO2. Conversely, despite the major presence of surface defects in the grey CeO2, the degradation percentage measured on grey CeO<sup>2</sup> after 3 h of reaction in the Fenton-like test (~10%) was lower compared to the degradation efficiency of the photocatalytic test (~16%) obtained with the same sample, which pointed to the slow regeneration of the Ce3<sup>+</sup> sites. As reported, the Fenton-like reaction with CeO<sup>2</sup> involves the formation of peroxide species on the surface of ceria due to the complexation of H2O<sup>2</sup> with Ce3<sup>+</sup> sites [25,30,58]. These peroxide species are chemically stable and can saturate the surface of CeO2, hindering the adsorption and subsequent oxidation of the organic target contaminant [25,30,58]. Indeed, a higher concentration of H2O<sup>2</sup> (superior to 3%, i.e., 0,9 M) both in the Fenton and in the photo-Fenton like reactions led to a considerable decrease in the catalytic activity of the grey CeO<sup>2</sup> (the highest performing sample for these tests, Figure S1). The regeneration and the further formation of Ce3<sup>+</sup> defect centres are, in this way, crucial steps. Interestingly, in the photo-Fenton like reaction (Figure 6c), the grey CeO<sup>2</sup> displayed the best performance (~35% of degradation), comparing all the investigated AOPs with the two CeO<sup>2</sup> samples. The solar irradiation could boost the further formation of Ce3<sup>+</sup> sites. An indirect confirmation is derived from the slight enhancement of the catalytic activity of bare CeO<sup>2</sup> (25%) compared to the solar photocatalytic test (20%) that can be attributed to the formation of in situ oxygen vacancies in the surface of bare CeO<sup>2</sup> which can react with the hydrogen peroxide. As reported in the literature [25,32], the irradiation of ceria with photons which possess energy higher than the CeO<sup>2</sup> band-gap can exploit the following reaction: $$\text{CaO}\_2 + \text{hv (E} \ge \text{E}\_\text{g}) \to \text{Ce}^{+3,+4}\text{O}\_{2\cdot x} + \text{x/2 }\text{O}\_2 \tag{9}$$ The interaction of the highly energetic photons with the surface of CeO<sup>2</sup> leads to the loss of surface oxygen, thus allowing the formation of the Ce3<sup>+</sup> states. The same reaction was exploited during the preparation of grey CeO2, where the formation of Ce3<sup>+</sup> was further increased due to the reducing atmosphere. Therefore, with the grey CeO2, owing to a higher number of defective centres compared to bare ceria (as shown by XPS and Raman analyses), it is possible to reach the best performance in the degradation of IMI by the photo-Fenton-like reaction. Furthermore, the contemporaneous presence of the hydrogen peroxide and the solar irradiation enhances the formation of hydroxyl radicals through the photolytic decomposition of H2O2, as confirmed by the experiment carried out without a catalyst (H2O<sup>2</sup> + IMI) that led to a slight variation in the initial concentration of IMI (Figure 6c, olive line). Catalysts 2020, 10, x FOR PEER REVIEW 9 of 16 formation of hydroxyl radicals through the photolytic decomposition of H2O2, as confirmed by the experiment carried out without a catalyst (H2O<sup>2</sup> + IMI) that led to a slight variation in the initial concentration of IMI (Figure 6c, olive line). It is important to highlight that, in the photocatalytic tests, the occurrence of the photon It is important to highlight that, in the photocatalytic tests, the occurrence of the photon interaction (reaction 9, reported above) can be exploited, but it has a minor role in determining the final performance. The presence of Ce3<sup>+</sup> was usually connected in the literature [59–61] to an improvement in the photocatalytic performance, especially under visible light irradiation, due to the presence of as-formed oxygen vacancies that shift the absorption of CeO<sup>2</sup> towards the visible-light region, improving the separation of the photogenerated charge carriers. However, the mean crystalline size and consequently the active surface area of the photocatalyst contribute considerably to the overall photocatalytic activity [34,62], as in our case, where the influence of the surface area is more preponderant than the effect of defects. For this reason, in the solar photocatalytic test, the bare CeO<sup>2</sup> (BET surface area of 81 m<sup>2</sup> /g) was more active than grey CeO<sup>2</sup> (BET surface area of 67 m<sup>2</sup> /g). interaction (reaction 9, reported above) can be exploited, but it has a minor role in determining the final performance. The presence of Ce3+ was usually connected in the literature [59–61] to an improvement in the photocatalytic performance, especially under visible light irradiation, due to the presence of as-formed oxygen vacancies that shift the absorption of CeO<sup>2</sup> towards the visible-light region, improving the separation of the photogenerated charge carriers. However, the mean crystalline size and consequently the active surface area of the photocatalyst contribute considerably to the overall photocatalytic activity [34,62], as in our case, where the influence of the surface area is more preponderant than the effect of defects. For this reason, in the solar photocatalytic test, the bare CeO<sup>2</sup> (BET surface area of 81 m2/g) was more active than grey CeO<sup>2</sup> (BET surface area of 67 m2/g). Finally, to test the reusability of grey CeO2, different photo-Fenton like reaction runs were performed on the same sample. In Figure 6d, the variation in the kinetic constant (referring to a first Finally, to test the reusability of grey CeO2, different photo-Fenton like reaction runs were performed on the same sample. In Figure 6d, the variation in the kinetic constant (referring to a first order kinetic [7] is reported with respect to the various runs. After five runs, the kinetic constant decreases from 12 ± 1·10−<sup>4</sup> min−<sup>1</sup> to 5.5 ± 0.6·10−<sup>4</sup> min−<sup>1</sup> , highlighting that the continuous redox Ce3+→Ce4<sup>+</sup> process on the surface of grey ceria led to a progressive deactivation of the catalyst, reasonably for the saturation of the surface sites with the products of IMI degradation. Nevertheless, when the same sample were pre-treated before the tests in the H<sup>2</sup> flow at room temperature for 1 h, it was possible to exploit an almost total reversibility of grey CeO<sup>2</sup> in the photo-Fenton-like reaction. In fact, the kinetic constant raised to 10 ± 1·10−<sup>4</sup> min−<sup>1</sup> in run 6 and went back to 5.0 ± 0.5·10−<sup>4</sup> min−<sup>1</sup> after the subsequent other four runs, pointing to the crucial role of H<sup>2</sup> in the restoring the Ce3<sup>+</sup> sites on grey CeO2. order kinetic [7] is reported with respect to the various runs. After five runs, the kinetic constant decreases from 12 ± 1·10−4 min−1 to 5.5 ± 0.6·10−4 min−1, highlighting that the continuous redox Ce3+→Ce4+ process on the surface of grey ceria led to a progressive deactivation of the catalyst, reasonably for the saturation of the surface sites with the products of IMI degradation. Nevertheless, when the same sample were pre-treated before the tests in the H<sup>2</sup> flow at room temperature for 1 h, it was possible to exploit an almost total reversibility of grey CeO<sup>2</sup> in the photo-Fenton-like reaction. In fact, the kinetic constant raised to 10 ± 1·10−4 min−1 in run 6 and went back to 5.0 ± 0.5·10−4 min−1 after the subsequent other four runs, pointing to the crucial role of H<sup>2</sup> in the restoring the Ce3+ sites on grey CeO2. The comparison with the commercial TiO<sup>2</sup> P25 Degussa (Figure 7) showed, as in the photocatalytic test, that bare CeO<sup>2</sup> had a comparable catalytic behaviour with respect to TiO<sup>2</sup> (Figure 7a), whereas this latter sample exhibited no substantial activity in the Fenton-like test (Figure 7b), The comparison with the commercial TiO<sup>2</sup> P25 Degussa (Figure 7) showed, as in the photocatalytic test, that bare CeO<sup>2</sup> had a comparable catalytic behaviour with respect to TiO<sup>2</sup> (Figure 7a), whereas this latter sample exhibited no substantial activity in the Fenton-like test (Figure 7b), and a lower activity compared to CeO<sup>2</sup> and grey CeO<sup>2</sup> in the photo-Fenton test (Figure 7c). This pointed out that, both for Fenton and photo-Fenton like reactions, the CeO2-based materials are better-performing than the commercial TiO2. As reported [63,64], the bare TiO<sup>2</sup> without structural (i.e., incorporation of surface defects) or chemical (as the formation of composites with iron oxides) modifications is not able to promote Fenton-like reactions. and a lower activity compared to CeO<sup>2</sup> and grey CeO2 in the photo-Fenton test (Figure 7c). This pointed out that, both for Fenton and photo-Fenton like reactions, the CeO2-based materials are better-performing than the commercial TiO2. As reported [63,64], the bare TiO<sup>2</sup> without structural (i.e., incorporation of surface defects) or chemical (as the formation of composites with iron oxides) modifications is not able to promote Fenton-like reactions. These data demonstrate the possibility of modifying and tuning the physico-chemical properties of CeO<sup>2</sup> with simple treatments, such as the solar light irradiation in a H<sup>2</sup> stream, so to maximize the catalytic performance. It is important to highlight, finally, that the CeO2 sample simply treated with H<sup>2</sup> or irradiated with a solar lamp without an H<sup>2</sup> stream did not show substantial changes compared to bare CeO<sup>2</sup> in the degradation performance of IMI in all the AOPs investigated. Figure 7. Cont. Catalysts 2020, 10, x FOR PEER REVIEW 10 of 16 Figure 7. (a) Photocatalytic degradation of IMI under solar light irradiation, (b) Fenton-like reaction, (c) photo-Fenton like reaction on the analyzed samples. Figure 7. (a) Photocatalytic degradation of IMI under solar light irradiation, (b) Fenton-like reaction, (c) photo-Fenton like reaction on the analyzed samples. 2.3. Tocixity Tests Artemia salina dehydrated cysts were employed for the acute toxicity test. Artemia salina nauplii can readily ingest fine particles smaller than 50 μm [65], and it is a nonselective filter-feeder organism. For these reasons, it is currently considered as a good model organism to assess in vivo nanoparticles toxicity, as previously demonstrated [66]. Low mortality percentages were evidenced after 24 and 48 hours of exposure (Table 1), at These data demonstrate the possibility of modifying and tuning the physico-chemical properties of CeO<sup>2</sup> with simple treatments, such as the solar light irradiation in a H<sup>2</sup> stream, so to maximize the catalytic performance. It is important to highlight, finally, that the CeO<sup>2</sup> sample simply treated with H<sup>2</sup> or irradiated with a solar lamp without an H<sup>2</sup> stream did not show substantial changes compared to bare CeO<sup>2</sup> in the degradation performance of IMI in all the AOPs investigated. different concentrations of both powders (bare CeO<sup>2</sup> and grey CeO2). Statistical analysis, carried out ### by one-way ANOVA test, gave no significant values for all the immobilization percentages nor 2.3. Tocixity Tests treated groups after 24 and 48 hours of exposure nor between treated and control (Ctrl, i.e., without metal oxide particles) groups (p > 0.05). The percentages of immobilized nauplii are reported in the Artemia salina dehydrated cysts were employed for the acute toxicity test. 3.3% (24 h) Grey CeO<sup>2</sup> Table 1. These data pointed to the low critical toxicity of the examined powders. Furthermore, it is possible to note that the modification of CeO<sup>2</sup> led to have a lower mortality with respect to the bare CeO2. Artemia salina nauplii can readily ingest fine particles smaller than 50 µm [65], and it is a non-selective filter-feeder organism. For these reasons, it is currently considered as a good model organism to assess in vivo nanoparticles toxicity, as previously demonstrated [66]. Figure 8 shows Artemia salina nauplii treated with bare CeO<sup>2</sup> for 24 hours, 48 hours and untreated nauplii (i.e., the controls). Table 1. Percentages of immobilized nauplii after exposition to CeO2 (bare CeO<sup>2</sup> and grey CeO2) at three different concentrations for 24 and 48 hours. Sample Ctrl 10-1 10-2 10-3 Bare CeO<sup>2</sup> 1.6% (24 h) 6.6% (48 h) 11.6% (24 h) 28.3% (48 h) 6.6% (24 h) 23.3% (48 h) 3.3% (24 h) 21.6% (48 h) Low mortality percentages were evidenced after 24 and 48 h of exposure (Table 1), at different concentrations of both powders (bare CeO<sup>2</sup> and grey CeO2). Statistical analysis, carried out by one-way ANOVA test, gave no significant values for all the immobilization percentages nor treated groups after 24 and 48 h of exposure nor between treated and control (Ctrl, i.e., without metal oxide particles) groups (p > 0.05). The percentages of immobilized nauplii are reported in the Table 1. These data pointed to the low critical toxicity of the examined powders. Furthermore, it is possible to note that the modification of CeO<sup>2</sup> led to have a lower mortality with respect to the bare CeO2. Sample Ctrl 10−<sup>1</sup> 10−<sup>2</sup> 10−<sup>3</sup> Bare CeO<sup>2</sup> 1.6% (24 h) 6.6% (48 h) 11.6% (24 h) 28.3% (48 h) 6.6% (24 h) 23.3% (48 h) 3.3% (24 h) 21.6% (48 h) Grey CeO<sup>2</sup> 3.3% (24 h) 8.3% (48 h) 8.3 % (24 h) 23.3% (48 h) 6.6% (24 h) 18.3% (48 h) 4.0% (24 h) 15.0% (48 h) 8.3% (48 h) 23.3% (48 h) 18.3% (48 h) 15.0% (48 h) Table 1. Percentages of immobilized nauplii after exposition to CeO<sup>2</sup> (bare CeO<sup>2</sup> and grey CeO<sup>2</sup> ) at three different concentrations for 24 and 48 h. 8.3 % (24 h) 6.6% (24 h) 4.0% (24 h) (a) (b) Figure 8 shows Artemia salina nauplii treated with bare CeO<sup>2</sup> for 24 h, 48 h and untreated nauplii (i.e., the controls). nauplii (i.e., the controls). CeO2. 2.3. Tocixity Tests (c) Figure 7. (a) Photocatalytic degradation of IMI under solar light irradiation, (b) Fenton-like reaction, Artemia salina nauplii can readily ingest fine particles smaller than 50 μm [65], and it is a nonselective filter-feeder organism. For these reasons, it is currently considered as a good model Low mortality percentages were evidenced after 24 and 48 hours of exposure (Table 1), at different concentrations of both powders (bare CeO<sup>2</sup> and grey CeO2). Statistical analysis, carried out by one-way ANOVA test, gave no significant values for all the immobilization percentages nor treated groups after 24 and 48 hours of exposure nor between treated and control (Ctrl, i.e., without metal oxide particles) groups (p > 0.05). The percentages of immobilized nauplii are reported in the Table 1. These data pointed to the low critical toxicity of the examined powders. Furthermore, it is possible to note that the modification of CeO<sup>2</sup> led to have a lower mortality with respect to the bare Figure 8 shows Artemia salina nauplii treated with bare CeO<sup>2</sup> for 24 hours, 48 hours and untreated Table 1. Percentages of immobilized nauplii after exposition to CeO2 (bare CeO<sup>2</sup> and grey CeO2) at Sample Ctrl 10-1 10-2 10-3 11.6% (24 h) 28.3% (48 h) 8.3 % (24 h) 23.3% (48 h) 6.6% (24 h) 23.3% (48 h) 6.6% (24 h) 18.3% (48 h) 3.3% (24 h) 21.6% (48 h) 4.0% (24 h) 15.0% (48 h) 11 of 16 Artemia salina dehydrated cysts were employed for the acute toxicity test. organism to assess in vivo nanoparticles toxicity, as previously demonstrated [66]. (c) photo-Fenton like reaction on the analyzed samples. three different concentrations for 24 and 48 hours. 1.6% (24 h) 6.6% (48 h) 3.3% (24 h) 8.3% (48 h) Bare CeO<sup>2</sup> Figure 8. Artemia salina nauplii: nauplii exposed to bare CeO<sup>2</sup> for 24 hours (a), nauplii exposed to bare CeO<sup>2</sup> for 48 hours (b), control at 24 hours (c), control at 48 hours (d). Figure 8. Artemia salina nauplii: nauplii exposed to bare CeO<sup>2</sup> for 24 h (a), nauplii exposed to bare CeO<sup>2</sup> for 48 h (b), control at 24 h (c), control at 48 h (d). ### 4. Materials and Methods 3. Materials and Methods ### 4.1. Samples Preparation 3.1. Samples Preparation The bare CeO<sup>2</sup> was prepared via chemical precipitation from Ce(NO3)3·6H2O (Fluka, Buchs, Switzerland) at pH > 8 utilizing a solution of KOH (1M, Fluka, Buchs, Switzerland). The obtained slurry was maintained under stirring at 80 °C for 3 h. After digestion for 24 h, it was filtered, washed with deionized water several times, and dried at 100 °C for 12 hours. Finally, the powders were calcined in air at 450 °C for 3 h. The modified CeO<sup>2</sup> (grey CeO2) was obtained with the same synthetic procedure reported above, but exposing the powders after calcination to the light of a solar lamp for 3 h (OSRAM Vitalux 300 W, 300-2000 nm; OSRAM Opto Semiconductors GmbH, Leibniz, Regensburg Germany) in a hydrogen stream (20 cc/min) at room temperature. The bare CeO<sup>2</sup> was prepared via chemical precipitation from Ce(NO3)3·6H2O (Fluka, Buchs, Switzerland) at pH > 8 utilizing a solution of KOH (1M, Fluka, Buchs, Switzerland). The obtained slurry was maintained under stirring at 80 ◦C for 3 h. After digestion for 24 h, it was filtered, washed with deionized water several times, and dried at 100 ◦C for 12 h. Finally, the powders were calcined in air at 450 ◦C for 3 h. The modified CeO<sup>2</sup> (grey CeO2) was obtained with the same synthetic procedure reported above, but exposing the powders after calcination to the light of a solar lamp for 3 h (OSRAM Vitalux 300 W, 300–2000 nm; OSRAM Opto Semiconductors GmbH, Leibniz, Regensburg Germany) in a hydrogen stream (20 cc/min) at room temperature. ### 4.2. Samples Characterization 3.2. Samples Characterization 4.3. (Photo)catalytic Experiments X-ray powder diffraction (XRD) measures were performed with a PANalytical X'pertPro X-ray diffractometer (Malvern PANalytical, Enigma Business Park, Grovewood Road, Malvern United Kingdom), employing a Cu Kα radiation. The identification of the crystalline phases was made comparing the diffractions with those of standard materials reported in the JCPDS Data File. Raman spectra were carried out with a WITec alpha 300 confocal Raman system (WITec Wissenschaftliche Instrumente und Technologie GmbH Ulm, Germany) with an excitation source at 532 nm under the same experimental condition reported in the ref. [67]. Fourier Transform Infrared Spectroscopy (FTIR) spectra were obtained in the range 4000–400 cm−1 using a Perkin Elmer FT-IR System 2000 (Perkin-Elmer, Waltham, MA, USA). The background spectrum was carried out with KBr. The textural properties of the samples were measured by N<sup>2</sup> adsorption–desorption at −196 °C with a Micromeritics Tristar II Plus 3020 (Micromeritics Instrument Corp. Norcross, USA), out-gassing the analysed materials at 100 °C overnight. UV-Vis-Diffuse Reflectance (UV-Vis DRS) spectra were obtained in the range of 200-800 nm using a Cary 60 spectrometer (Agilent Stevens Creek Blvd. Santa Clara, United States). X-ray photoelectron spectroscopy (XPS) measurements were recorded using a K-Alpha X-ray photoelectron spectrometer (Thermo Fisher Scientific Waltham, MA USA), utilizing the C 1 peak at 284.9 eV (ascribed to adventitious carbon) as a reference. X-ray powder diffraction (XRD) measures were performed with a PANalytical X'pertPro X-ray diffractometer (Malvern PANalytical, Enigma Business Park, Grovewood Road, Malvern United Kingdom), employing a Cu Kα radiation. The identification of the crystalline phases was made comparing the diffractions with those of standard materials reported in the JCPDS Data File. Raman spectra were carried out with a WITec alpha 300 confocal Raman system (WITec Wissenschaftliche Instrumente und Technologie GmbH Ulm, Germany) with an excitation source at 532 nm under the same experimental condition reported in the ref. [67]. Fourier Transform Infrared Spectroscopy (FTIR) spectra were obtained in the range 4000–400 cm−<sup>1</sup> using a Perkin Elmer FT-IR System 2000 (Perkin-Elmer, Waltham, MA, USA). The background spectrum was carried out with KBr. The textural properties of the samples were measured by N<sup>2</sup> adsorption–desorption at <sup>−</sup><sup>196</sup> ◦C with a Micromeritics Tristar II Plus 3020 (Micromeritics Instrument Corp. Norcross, USA), out-gassing the analysed materials at 100 ◦C overnight. UV-Vis-Diffuse Reflectance (UV-Vis DRS) spectra were obtained in the range of 200–800 nm using a Cary 60 spectrometer (Agilent Stevens Creek Blvd. Santa Clara, United States). X-ray photoelectron spectroscopy (XPS) measurements were recorded using a K-Alpha X-ray photoelectron spectrometer (Thermo Fisher Scientific Waltham, MA USA), utilizing the C 1 peak at 284.9 eV (ascribed to adventitious carbon) as a reference. stirred for 120 min in the dark so as to achieve the adsorption/desorption equilibrium. During the tests, aliquots of the suspension were withdrawn at a given time interval to measure the IMI concentration by means of Cary 60 UV–vis spectrophotometer (Agilent Stevens Creek Blvd. Santa Clara, United States). The IMI degradation was evaluated by following the absorbance peaks at 270 nm in the Lambert–Beer regime, reporting the C/C<sup>0</sup> ratio as a function of time t, where C is the concentration of the contaminant at the time t, while C<sup>0</sup> is the starting concentration of the pollutant. The photocatalytic tests were performed utilizing a solar lamp (OSRAM Vitalux 300 W, 300-2000	doab	2025-04-07T03:56:58.838694	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 116 }
004db1df-dc76-42af-952f-1f5e82b7822b.117	3.3. (Photo)catalytic Experiments The photocatalytic tests were performed utilizing a solar lamp (OSRAM Vitalux 300 W, 300–2000 nm, OSRAM Opto Semiconductors GmbH, Leibniz, Regensburg Germany) irradiating a jacketed Pyrex batch reactor, kept at 25 ◦C. A total of 50 mg of powder was suspended in 50 mL of the reactant solution containing 5×10−<sup>5</sup> M of IMI (Sigma-Aldrich, Buchs, Switzerland). The reaction mixture was stirred for 120 min in the dark so as to achieve the adsorption/desorption equilibrium. During the tests, aliquots of the suspension were withdrawn at a given time interval to measure the IMI concentration by means of Cary 60 UV–vis spectrophotometer (Agilent Stevens Creek Blvd. Santa Clara, United States). The IMI degradation was evaluated by following the absorbance peaks at 270 nm in the Lambert–Beer regime, reporting the C/C<sup>0</sup> ratio as a function of time t, where C is the concentration of the contaminant at the time t, while C<sup>0</sup> is the starting concentration of the pollutant. The Fenton-like reaction was carried out with the same apparatus described above, adding 5 mL of hydrogen peroxide (3%, 0.9 M Fluka, Buchs, Switzerland) in the reactor without irradiation; in the photo-Fenton-like tests the solar light irradiation was employed, too. In all the catalytic tests the experimental error was 1%, i.e., within the symbol size.	doab	2025-04-07T03:56:58.841016	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 117 }
004db1df-dc76-42af-952f-1f5e82b7822b.118	3.4. Toxicity Tests Artemia salina dehydrated cysts were used for the acute toxicity test. Cysts (Hobby, Germany) were hydrated in ASPM seawater solution (ASPM is an artificial seawater made of: NaCl = 26.4 g, KCl = 0.84 g, CaCl2·H2O = 1.67 g, MgCl·H2O = 4.6 g, MgSO4·7H2O = 5.58 g, NaHCO<sup>3</sup> = 0.17 g, and H3BO<sup>3</sup> = 0.03 g) maintaining standard laboratory conditions (1.500 lux daylight; 26 ± 1 ◦C; continuous aeration), then nauplii hatched within 24 h. Two stock solutions of CeO<sup>2</sup> (bare CeO<sup>2</sup> and grey CeO2) were prepared after dilution in ASPM solution. Then, fresh suspensions with different concentrations of powders (10−<sup>1</sup> , 10−<sup>2</sup> , 10−<sup>3</sup> mg/mL) were made starting from the stock suspensions (1 mg/10 mL). These solutions were vortexed for 30 s. One nauplius per well in 96-well microplates, was added with 200 µL of each different concentration of powder solutions. They were incubated at 26 ◦C for 24/48 h. The number of surviving nauplii in each well was counted under a stereomicroscope after 24/48 h. A control group was also set up with ASPM seawater solution only. Larvae were not fed during the bioassays. At the end of the test, the endpoints (immobility, i.e., death) were evaluated with a stereomicroscope (Leica EZ4, Leica Microsystems Srl, Buccinasco (MI), Italy): a nauplium was considered to be immobile or dead if it could not move its antennae after slight agitation of the water for 10 seconds. Larvae that were completely motionless were counted as dead, and the percentages of mortality compared to the control were calculated. The death % of the crustacean for each concentration was calculated as follows: (n. dead nauplii/n. total animal treated 100). Data were analyzed for differences between the control and treatments using one-way ANOVA followed by Tukey's test, where p < 0.05 is considered significant and p < 0.01 extremely significant.	doab	2025-04-07T03:56:58.841120	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 118 }
004db1df-dc76-42af-952f-1f5e82b7822b.119	4. Conclusions Different AOPs (photocatalysis, Fenton and photo-Fenton reactions) were investigated for the degradation of the IMI insecticide. Two CeO<sup>2</sup> samples were tested, one prepared with the conventional precipitation route and another one modifying the as-prepared CeO<sup>2</sup> by exposing it to solar light irradiation in a hydrogen stream. The latter treatment allowed to obtain a more defective ceria with increased performance in the solar photo-Fenton reaction. This hybrid AOP obtained the best degradation rate of the IMI. The photon interaction with the surface of CeO<sup>2</sup> led to a loss of oxygen with the formation of Ce3<sup>+</sup> centers, which is essential to boost the degradation rate of the pesticide degradation through the photo-Fenton process. The solar irradiation in a reducing atmosphere could obtain a defective ceria that can be exploited to explore new synergisms between different AOPs for wastewater purification over non-conventional photocatalysts/Fenton materials. Furthermore, the investigated materials did not exhibit critical toxicity. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/4/446/s1, Figure S1: Influence of the H2O<sup>2</sup> concentration. Author Contributions: Conceptualization R.F. and G.I.; Catalytic tests R.F. and S.A.B.; Investigations and writing R.F.; Raman measurements L.D.; Toxicity tests M.V.B.; R.P. and E.M.S.; Review and editing G.I., S.S. and V.P. All authors have read and agreed to the current version of the manuscript. Funding: This work was partially founded by the Micro WatTS project, Interreg V. A. Italia-Malta (CUP: B61G18000070009). R.F. thanks the PON project "AIM" founded by the European Social Found (ESF) CUP: E66C18001220005 for the financial support. R.P. thanks the PON project "AIM" founded by the European Social Found (ESF) CUP: E66C18001300007 for the financial support. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.841264	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 119 }
004db1df-dc76-42af-952f-1f5e82b7822b.121	1. Introduction Since the industrial revolution, the development of various industries has made life in human society more convenient, but has also caused many environmental problems. Wastewater, such as cooling water and clean water for equipment, is discharged from various industrial processes. The constituents in any wastewater are diverse and complex consisting of raw materials, intermediate products, by-products, and end products. Charging these compounds directly to the environment can have detrimental consequences. For example, oxygen-containing organic compounds such as aldehydes, ketones, and ethers are reductive, meaning that they are capable of consuming dissolved oxygen in the water to a low-level endangering aquatic organisms. Wastewater can also contain a large amount of nitrogen, phosphorus, and potassium which can promote the growth of algae and triggering eutrophication pollution in water bodies [1]. Released toxic substances from wastewater can bio-accumulate in fish and eventually pass to people who consumed it. Thus, wastewater treatment is an important process to avoid these consequences. Within the treatment options, physical, biological, and chemical methods are mainly used to treat wastewater by removing pollutants in the water and reducing organic pollutants and eutrophic substances in the water [2–8]. Among many treatment processes, the in-site chemical oxidation method is to inject and mix oxidants into the underground environment aiming to degrade pollutants Citation: Wen, P.-Y.; Lai, T.-Y.; Wu, T.; Lin, Y.-W. Hydrothermal and Co-Precipitated Synthesis of Chalcopyrite for Fenton-like Degradation toward Rhodamine B. Catalysts 2022, 12, 152. https:// doi.org/10.3390/catal12020152 Academic Editor: Roberto Fiorenza Received: 31 December 2021 Accepted: 25 January 2022 Published: 26 January 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). in groundwater and soil [9–11]. Under ideal conditions, this chemical treatment can convert organic pollutants into less toxic molecules, such as carbon dioxide, water, and inorganic salts. The commonly used oxidants are permanganate (MnO<sup>4</sup> −), Fenton reagent (Fe2+/H2O2), and ozone (O3) [12–15]. The mechanism of Fenton reagent (Fe2+/H2O2) in reactions has been known to produce •OH radicals, which can cleave C–H bonds of organic compounds, turning them into environmentally benign final products [16,17]. However, this Fenton method has some drawbacks including the specific pH working range and Fe sludge precipitation at the end of the Fenton reaction [18,19]. Another alternative way to generate radicals with a wider pH working range is by using persulfate salts [20–26]. Persulfate salts can come from two types: peroxymonosulfate (HSO<sup>5</sup> −) and peroxydisulfate (S2O<sup>8</sup> <sup>2</sup>−), both of which contain an O–O bond (peroxide group) capable of generating •SO<sup>4</sup> <sup>−</sup> radicals and •OH radicals in Fenton-like reaction for degradation of organic compounds [27,28]. Persulfate salts are strong oxidant (E<sup>0</sup> = 2.1 V), yet they are very stable for transportation and prolong storage making them very attractive oxidants for underground water treatment [29]. There are a few ways of activating persulfate to generate radicals, such as thermal decomposition, alkaline activation, transition metal ions activation, and heterogeneous catalysis [30]. Among them all, transition metal ions activation is considered the simplest and most benign method with no external energy requirement and recyclability of transition metal ions [31–34]. Cobalt ions are commonly used in activating persulfate in research, but their hazardous nature makes them unsuitable for water treatment [35,36]. Thus, it is necessary to find an alternative transition metal catalyst that can be used in water treatment. Recently, Cu/Fe-bearing solids such as chalcopyrite (CuFeS2) have been widely used as catalysts in advanced oxidation processes (AOPs) for wastewater treatment [37–41]. For instance, Dotto et al. demonstrated the ability of their prepared citrate-CuFeS<sup>2</sup> materials to degrade 90% of bisphenol A (BPA) in a 15-min Fenton process [42]. Their novel CuFeS<sup>2</sup> samples were prepared with a microwave reactor (1400 W, 200 ◦C, 7 min). Pastrana-Martinez et al. used the mineral of CuFeS<sup>2</sup> mined from Jendouba, Tunisia, to catalyze tyrosol degradation (85.0% degradation within 60 min) by using a UV light-assisted Fenton reaction [43]. However, for ground water treatment, this method requires UV light as external energy requirement. Chang et al. proposed that the microwave-assisted synthesis of CuFeS2/Ag3PO<sup>4</sup> with enhanced rhodamine B (RhB) degradation (96% degradation within 1 min) under visible light-Fenton process [44]. However, these methods also need light irradiation to improve the degradation performance of CuFeS2. Herein, we synthesized CuFeS<sup>2</sup> samples through hydrothermal and co-precipitated method to realize the advantages in material preparation, stability of materials, and degradation performance in new water treatment option. We expected higher temperature and pressure treatment (hydrothermal process) to make the prepared particles with small size and high special surface area compared to the co-precipitated process, resulting in higher catalytic activity [45–47]. In order to prove this, the prepared CuFeS<sup>2</sup> samples in the presence of Na2S2O<sup>8</sup> were used to evaluate the degradation efficiency of various dyestuff (RhB, rhodamine 6G [R6G], methylene blue [MB], methyl orange [MO], and BPA). The degradation mechanism of CuFeS<sup>2</sup> was elucidated and the reactive species were identified. Finally, the practical applications of CuFeS<sup>2</sup> samples in the treatment of environmental samples were demonstrated.	doab	2025-04-07T03:56:58.841400	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 121 }
004db1df-dc76-42af-952f-1f5e82b7822b.123	2.1. Characterization of the CuFeS<sup>2</sup> Samples The morphology and composition of the prepared H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> samples were analyzed through SEM and EDS-mapping (Figure 1). As shown in Figure 1, H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> samples appear as sphere-like structures, with the average diameter ranging 25–40 nm and 95–125 nm, respectively. The smaller particle size of the H-CuFeS<sup>2</sup> samples can be the result of hydrothermal treatment which hindered the particle growth. On the other hand, high concentration of N2H4·H2O was used as reducing agent to prepare C-CuFeS<sup>2</sup> samples, resulting in particle agglomeration. In addition, Ostwald ripening may occur during heating procedure. Therefore, small C-CuFeS<sup>2</sup> samples dissolved and redeposited onto larger C-CuFeS<sup>2</sup> samples. From the results of energy dispersive spectrometer (EDS)-mapping (green color, S elements; blue color, Fe elements; and red color, Cu elements), the presence of Cu, Fe, and S elements in both CuFeS<sup>2</sup> samples were confirmed and dispersed well in their crystals. C-CuFeS2 samples, resulting in particle agglomeration. In addition, Ostwald ripening may occur during heating procedure. Therefore, small C-CuFeS2 samples dissolved and redeposited onto larger C-CuFeS2 samples. From the results of energy dispersive spectrometer (EDS)-mapping (green color, S elements; blue color, Fe elements; and red color, Cu elements), the presence of Cu, Fe, and S elements in both CuFeS2 samples were confirmed and dispersed well in their crystals. CuFeS2 and C-CuFeS2 samples appear as sphere-like structures, with the average diameter ranging 25–40 nm and 95–125 nm, respectively. The smaller particle size of the H-CuFeS2 samples can be the result of hydrothermal treatment which hindered the particle growth. On the other hand, high concentration of N2H4·H2O was used as reducing agent to prepare Catalysts 2022, 11, x FOR PEER REVIEW 3 of 21 Figure 1. SEM images and EDS-mapping of (A) H-CuFeS2 and (B) C-CuFeS2 samples. Figure 1. SEM images and EDS-mapping of (A) H-CuFeS<sup>2</sup> and (B) C-CuFeS<sup>2</sup> samples. Figures 2 and 3 showed the TEM images and EDS spectra of the CuFeS2 samples. The diameters of both CuFeS2 samples from TEM images were consistent with the SEM results. We also found both CuFeS2 samples possessed 0.31 nm and 0.23 nm of lattice lines, corresponding to the crystal planes of (112) and (204). The EDS spectra of the prepared CuFeS2 samples confirm the presence of Cu, Fe, and S elements in their crystals, accordingly. The atomic ratios (Cu:Fe:S) for the H-CuFeS2 and C-CuFeS2 samples were determined to be 1.1:1:1.8 and 1.4:1:2.0, respectively. High content of Cu elements in the C-CuFeS2 in the sample is consistent with lower solubility predicted from smaller Ksp value of Cu2S when comparing with Fe2S3 (Ksp of Fe2S3: 3.7 × 10−19, Ksp of Cu2S: 2.0 × 10−47). Figures 2 and 3 showed the TEM images and EDS spectra of the CuFeS<sup>2</sup> samples. The diameters of both CuFeS<sup>2</sup> samples from TEM images were consistent with the SEM results. We also found both CuFeS<sup>2</sup> samples possessed 0.31 nm and 0.23 nm of lattice lines, corresponding to the crystal planes of (112) and (204). The EDS spectra of the prepared CuFeS<sup>2</sup> samples confirm the presence of Cu, Fe, and S elements in their crystals, accordingly. The atomic ratios (Cu:Fe:S) for the H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> samples were determined to be 1.1:1:1.8 and 1.4:1:2.0, respectively. High content of Cu elements in the C-CuFeS<sup>2</sup> in the sample is consistent with lower solubility predicted from smaller Ksp value of Cu2S when comparing with Fe2S<sup>3</sup> (Ksp of Fe2S3: 3.7 <sup>×</sup> <sup>10</sup>−19, Ksp of Cu2S: 2.0 <sup>×</sup> <sup>10</sup>−47). XRD was used to investigate the crystal structure of the prepared CuFeS<sup>2</sup> samples. The XRD patterns of the prepared CuFeS<sup>2</sup> samples are shown in Figure 4A. The diffraction peaks at 29.5◦ , 49.1◦ , and 58.6◦ were identified and assigned to the (112), (204), (312), (204), and (312) faces of the tetragonal chalcopyrite CuFeS2, respectively (PDF 83-0983). Through the Scherrer equation, the average crystal size of H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> was 20.36 and 11.2 nm, respectively. The Raman spectra of the prepared CuFeS<sup>2</sup> samples are shown in Figure 4B. The Raman shifts at 212 cm−<sup>1</sup> , 276 cm−<sup>1</sup> , and 379 cm−<sup>1</sup> correspond to the S element, Cu(I)-S, and Fe(III)-S stretching vibration, respectively. Catalysts 2022, 11, x FOR PEER REVIEW 4 of 21 Figure 2. TEM images and EDS spectra of H-CuFeS Figure 2. TEM images and EDS spectra of H-CuFeS 2 2 samples. samples. Catalysts 2022, 11, x FOR PEER REVIEW 5 of 21 XRD was used to investigate the crystal structure of the prepared CuFeS2 samples. and (312) faces of the tetragonal chalcopyrite CuFeS2, respectively (PDF 83-0983). Through the Scherrer equation, the average crystal size of H-CuFeS2 and C-CuFeS2 was 20.36 and 11.2 nm, respectively. The Raman spectra of the prepared CuFeS2 samples are shown in Figure 4B. The Raman shifts at 212 cm−1, 276 cm−1, and 379 cm−1 correspond to the S ele- Figure 3. TEM images and EDS spectra of C-CuFeS2 samples. Figure 3. TEM images and EDS spectra of C-CuFeS<sup>2</sup> samples. ment, Cu(I)-S, and Fe(III)-S stretching vibration, respectively. Figure 4. (A) XRD and (B) Raman spectra of H-CuFeS2 (black), and C-CuFeS2 (red) samples. Figure 4. (A) XRD and (B) Raman spectra of H-CuFeS<sup>2</sup> (black), and C-CuFeS<sup>2</sup> (red) samples. As another quality assurance method, XPS analysis of the prepared CuFeS2 samples (Figures 5 and 6) revealed that it contains three elements: Cu, Fe, and S [48,49]. Highresolution XPS revealed Cu2p, Fe2p, and S2p in the H-CuFeS2 samples as shown in Figures 5B, 5C, and 5D, respectively. In Figure 5B, the peaks at 931.9 and 951.7 eV correspond to Cu+ 2p3/2 and Cu+ 2p1/2, respectively, whereas those at 933.2 and 953.0 eV correspond to Cu2+ 2p3/2 and Cu2+ 2p1/2, respectively. The peaks at 711.7 and 724.9 eV correspond to Fe2+ 2p3/2 and Fe2+ 2p1/2, respectively, whereas those at 715.2 and 734.3 eV correspond to Fe3+ 2p3/2 and Fe3+ 2p1/2, respectively (Figure 5C). The peaks at 162.5 and 167.8 eV correspond to S2− 2p and S6+ 2p, respectively (Figure 5D). For C-CuFeS2 samples, the peaks at 931.9 and 951.6 eV correspond to Cu+ 2p3/2 and Cu+ 2p1/2, respectively, whereas those at 934.1 and 952.7 eV correspond to Cu2+ 2p3/2 and Cu2+ 2p1/2, respectively (Figure 6B). The peaks at 711.4 As another quality assurance method, XPS analysis of the prepared CuFeS<sup>2</sup> samples (Figures 5 and 6) revealed that it contains three elements: Cu, Fe, and S [48,49]. Highresolution XPS revealed Cu2p, Fe2p, and S2p in the H-CuFeS<sup>2</sup> samples as shown in Figure 5B–D, respectively. In Figure 5B, the peaks at 931.9 and 951.7 eV correspond to Cu<sup>+</sup> 2p3/2 and Cu<sup>+</sup> 2p1/2, respectively, whereas those at 933.2 and 953.0 eV correspond to Cu2+ 2p3/2 and Cu2+ 2p1/2, respectively. The peaks at 711.7 and 724.9 eV correspond to Fe2+ 2p3/2 and Fe2+ 2p1/2, respectively, whereas those at 715.2 and 734.3 eV correspond to Fe3+ 2p3/2 and Fe3+ 2p1/2, respectively (Figure 5C). The peaks at 162.5 and 167.8 eV correspond to S2<sup>−</sup> 2p and S6+ 2p, respectively (Figure 5D). For C-CuFeS<sup>2</sup> samples, the peaks at 931.9 and 951.6 eV correspond to Cu<sup>+</sup> 2p3/2 and Cu<sup>+</sup> 2p1/2, respectively, whereas those at 934.1 and 952.7 eV correspond to Cu2+ 2p3/2 and Cu2+ 2p1/2, respectively (Figure 6B). The peaks at 711.4 and 724.8 eV correspond to Fe2+ 2p3/2 and Fe2+ 2p1/2, respectively, whereas those at 714.6 and 734.2 eV correspond to Fe3+ 2p3/2 and Fe3+ 2p1/2, respectively (Figure 6C). The peaks at 162.5 and 168.9 eV correspond to S2<sup>−</sup> 2p and S6+ 2p, respectively (Figure 6D). and 724.8 eV correspond to Fe2+ 2p3/2 and Fe2+ 2p1/2, respectively, whereas those at 714.6 and 734.2 eV correspond to Fe3+ 2p3/2 and Fe3+ 2p1/2, respectively (Figure 6C). The peaks at 162.5 and 168.9 eV correspond to S2− 2p and S6+ 2p, respectively (Figure 6D). According to its peak area, the percentage of different oxidation states of each element in the prepared CuFeS2 samples can be estimated. In H-CuFeS2 samples, elemental According to its peak area, the percentage of different oxidation states of each element in the prepared CuFeS<sup>2</sup> samples can be estimated. In H-CuFeS<sup>2</sup> samples, elemental compositions were found 82.3% Cu<sup>+</sup> and 17.6% Cu2+ from Cu analysis, 66.9% Fe2+ and 33.1% Fe 3+ from Fe analysis, and 74.2% S2<sup>−</sup> and 25.7% S6+ from sulfur analysis. In C-CuFeS<sup>2</sup> samples, elemental composition was found to be 90.6% Cu<sup>+</sup> vs. 9.3% Cu2+ for Cu, 60.8% Fe2+ vs. 39.2% Fe 3+ for Fe, and 62.6% S2<sup>−</sup> vs. 37.3%. S6+ for S. compositions were found 82.3% Cu+ and 17.6% Cu2+ from Cu analysis, 66.9% Fe2+ and 33.1% Fe 3+ from Fe analysis, and 74.2% S2− and 25.7% S6+ from sulfur analysis. In C-CuFeS2 samples, elemental composition was found to be 90.6% Cu+ vs. 9.3% Cu2+ for Cu, 60.8% Fe2+ vs. 39.2% Fe 3+ for Fe, and 62.6% S2− vs. 37.3%. S6+ for S. Figure 5. XPS spectra of H-CuFeS2 samples: (A) full scan, (B) Cu2p, (C) Fe2p, and (D) S2p. Figure 5. XPS spectra of H-CuFeS<sup>2</sup> samples: (A) full scan, (B) Cu2p, (C) Fe2p, and (D) S2p. Figure 5. XPS spectra of H-CuFeS2 samples: (A) full scan, (B) Cu2p, (C) Fe2p, and (D) S2p. Figure 6. XPS spectra of C-CuFeS2 samples: (A) full scan, (B) Cu2p, (C) Fe2p, and (D) S2p. Figure 6. Figure 6. XPS spectra of C-CuFeS XPS spectra of C-CuFeS<sup>2</sup> samples: ( 2 samples: (AA) full scan, ( ) full scan, (BB) Cu) Cu2p2p, (, (CC) Fe ) Fe2p2p, and ( , and (DD) S) S2p2p. . ## 2.2. Degradation Performance of the CuFeS<sup>2</sup> Samples Catalysts 2022, 11, x FOR PEER REVIEW 8 of 21 The degradation activity of the prepared CuFeS<sup>2</sup> samples was evaluated with RhB (20 ppm) first. According to our previous experience, the degradation efficiency decreased with an increasing dye concentration. This is because the excessive coverage of dye on the active surface of catalysts leads to a decrease in the catalytic activity. Thus, 20 ppm RhB was selected for the experiment. The variations in the RhB concentration (C/C0), where C<sup>0</sup> is the initial RhB concentration, and C is the RhB concentration at time t, with the reaction time for the prepared CuFeS<sup>2</sup> samples in the presence of H2O<sup>2</sup> (Fenton reaction) and Na2S2O<sup>8</sup> (Fenton-like reaction), were found in Figure 7. Prior to the addition of the oxidant, each catalyst (0.20 g) was introduced to the 20 ppm RhB solution for 30 min in the dark (indicated as "−30 min" in Figure 7) to reach equilibrium. The RhB concentration for H-CuFeS<sup>2</sup> samples after this equilibration time is lower than that of C-CuFeS<sup>2</sup> samples, reflecting RhB adsorption on H-CuFeS<sup>2</sup> samples. This is because smaller size of the H-CuFeS<sup>2</sup> samples had higher specific surface area than C-CuFeS<sup>2</sup> samples. Through the Fenton reaction, the degradation efficiency within 30 min was 32.3% and 26.4% for the H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> samples, respectively (black and blue curve). This suggests that the degradation performance of H-CuFeS<sup>2</sup> is better than that of C-CuFeS2, attributable to adsorption ability of high specific surface area for the H-CuFeS<sup>2</sup> samples. The results of RhB degradation through a Fenton-like reaction by the H-CuFeS<sup>2</sup> and C-CuFeS<sup>2</sup> samples were shown in the red and pink curve. The degradation efficiency within 30 min reaction time follows this order: H-CuFeS<sup>2</sup> (93.7%) > C-CuFeS<sup>2</sup> (66.3%), indicating H-CuFeS<sup>2</sup> having higher catalytic activity to produce •SO<sup>4</sup> − radicals. Furthermore, we found that degradation performance of •SO<sup>4</sup> <sup>−</sup> radicals is higher than that of •OH radicals for both CuFeS<sup>2</sup> samples. This is because of the different lifetimes of radicals (•SO<sup>4</sup> − radicals: 4 s, •OH radicals: 1 µs). Thus, the degradation system of H-CuFeS<sup>2</sup> through a Fenton-like reaction was selected for the further study. 2.2. Degradation Performance of the CuFeS2 Samples The degradation activity of the prepared CuFeS2 samples was evaluated with RhB (20 ppm) first. According to our previous experience, the degradation efficiency decreased with an increasing dye concentration. This is because the excessive coverage of dye on the active surface of catalysts leads to a decrease in the catalytic activity. Thus, 20 ppm RhB was selected for the experiment. The variations in the RhB concentration (C/C0), where C0 is the initial RhB concentration, and C is the RhB concentration at time t, with the reaction time for the prepared CuFeS2 samples in the presence of H2O2 (Fenton reaction) and Na2S2O8 (Fenton-like reaction), were found in Figure 7. Prior to the addition of the oxidant, each catalyst (0.20 g) was introduced to the 20 ppm RhB solution for 30 min in the dark (indicated as "−30 min" in Figure 7) to reach equilibrium. The RhB concentration for H-CuFeS2 samples after this equilibration time is lower than that of C-CuFeS2 samples, reflecting RhB adsorption on H-CuFeS2 samples. This is because smaller size of the H-CuFeS2 samples had higher specific surface area than C-CuFeS2 samples. Through the Fenton reaction, the degradation efficiency within 30 min was 32.3% and 26.4% for the H-CuFeS2 and C-CuFeS2 samples, respectively (black and blue curve). This suggests that the degradation performance of H-CuFeS2 is better than that of C-CuFeS2, attributable to adsorption ability of high specific surface area for the H-CuFeS2 samples. The results of RhB degradation through a Fenton-like reaction by the H-CuFeS2 and C-CuFeS2 samples were shown in the red and pink curve. The degradation efficiency within 30 min reaction time follows this order: H-CuFeS2 (93.7%) > C-CuFeS2 (66.3%), indicating H-CuFeS2 having higher catalytic activity to produce •SO4− radicals. Furthermore, we found that degradation performance of •SO4− radicals is higher than that of •OH radicals for both CuFeS2 samples. This is because of the different lifetimes of radicals (•SO4− radicals: 4 s, •OH radicals: 1 μs). Thus, the degradation system of H-CuFeS2 through a Fenton-like reaction was selected for the further study. Figure 7. Fenton and Fenton-like reactions for RhB degradation at different conditions: H-CuFeS2 in the presence of H2O2 (black), H-CuFeS2 in the presence of Na2S2O8 (red), C-CuFeS2 in the presence of H2O2 (blue), and C-CuFeS2 in the presence of Na2S2O8 (pink). Figure 7. Fenton and Fenton-like reactions for RhB degradation at different conditions: H-CuFeS<sup>2</sup> in the presence of H2O<sup>2</sup> (black), H-CuFeS<sup>2</sup> in the presence of Na2S2O<sup>8</sup> (red), C-CuFeS<sup>2</sup> in the presence of H2O<sup>2</sup> (blue), and C-CuFeS<sup>2</sup> in the presence of Na2S2O<sup>8</sup> (pink). To maximize the degradation performance of H-CuFeS2, the effect from various concentrations of Na2S2O<sup>8</sup> was studied. As shown in Figure 8, the degradation efficiency increased with increasing Na2S2O<sup>8</sup> concentration. Due to low solubility of Na2S2O8, we selected 4.0 mM of Na2S2O<sup>8</sup> as the optimum required concentration of Na2S2O8. Dye adsorption on H-CuFeS<sup>2</sup> was observed in the absence of Na2S2O<sup>8</sup> (black cure in Figure 9). Although direct degradation of RhB by Na2S2O<sup>8</sup> without H-CuFeS<sup>2</sup> was noticed from the experiment due to the high oxidizing strength of Na2S2O<sup>8</sup> (red curve in Figure 9), its rate of degradation cannot compete with H-CuFeS<sup>2</sup> samples in the presence of Na2S2O8, which achieved an impressive 98.8% within 10 min (blue cure in Figure 9). In addition, we also analyzed the degradation performances of Cu2S and FeS<sup>2</sup> nanoparticles to investigate which element is important for a Fenton-like reaction. As shown in Figure 10, the RhB degradation efficiency within 15 min reaches 64.1% and 89.0% for Cu2S and FeS nanoparticles, respectively. These results suggested that the FeS<sup>2</sup> nanoparticles catalyze Na2S2O<sup>8</sup> to produce •SO<sup>4</sup> − radicals better than Cu2S nanoparticles, indicating Fe component is important than Cu component for the Fenton-like reaction. hough direct degradation of RhB by Na2S2O8 without H-CuFeS2 was noticed from the experiment due to the high oxidizing strength of Na2S2O8 (red curve in Figure 9), its rate of degradation cannot compete with H-CuFeS2 samples in the presence of Na2S2O8, which achieved an impressive 98.8% within 10 min (blue cure in Figure 9). In addition, we also analyzed the degradation performances of Cu2S and FeS2 nanoparticles to investigate which element is important for a Fenton-like reaction. As shown in Figure 10, the RhB degradation efficiency within 15 min reaches 64.1% and 89.0% for Cu2S and FeS nanoparticles, respectively. These results suggested that the FeS2 nanoparticles catalyze Na2S2O8 to produce •SO4− radicals better than Cu2S nanoparticles, indicating Fe component is important than Cu component for the Fenton-like reaction. To maximize the degradation performance of H-CuFeS2, the effect from various concentrations of Na2S2O8 was studied. As shown in Figure 8, the degradation efficiency increased with increasing Na2S2O8 concentration. Due to low solubility of Na2S2O8, we selected 4.0 mM of Na2S2O8 as the optimum required concentration of Na2S2O8. Dye adsorption on H-CuFeS2 was observed in the absence of Na2S2O8 (black cure in Figure 9). Alt- Catalysts 2022, 11, x FOR PEER REVIEW 9 of 21 Figure 8. Fenton-like reaction for RhB degradation by H-CuFeS2 samples at different concentration of Na2S2O8. Figure 8. Fenton-like reaction for RhB degradation by H-CuFeS<sup>2</sup> samples at different concentration of Na2S2O<sup>8</sup> . Catalysts 2022, 11, x FOR PEER REVIEW 10 of 21 Figure 9. Fenton-like reaction for RhB degradation under different conditions: H-CuFeS2 samples only (black), Na2S2O8 only (red), and H-CuFeS2 in the presence of Na2S2O8 (blue). Top image: photographs of the RhB solution under the Fenton reaction at different reaction time. Figure 9. Fenton-like reaction for RhB degradation under different conditions: H-CuFeS<sup>2</sup> samples only (black), Na2S2O<sup>8</sup> only (red), and H-CuFeS<sup>2</sup> in the presence of Na2S2O<sup>8</sup> (blue). Top image: photographs of the RhB solution under the Fenton reaction at different reaction time. Figure 10. Fenton-like reaction for RhB degradation in the presence of Na2S2O8 by using different catalysts: Cu2S (black), FeS2 (red), and H-CuFeS2 (blue). Figure 9. Fenton-like reaction for RhB degradation under different conditions: H-CuFeS2 samples only (black), Na2S2O8 only (red), and H-CuFeS2 in the presence of Na2S2O8 (blue). Top image: pho- tographs of the RhB solution under the Fenton reaction at different reaction time. Figure 10. Fenton-like reaction for RhB degradation in the presence of Na2S2O8 by using different catalysts: Cu2S (black), FeS2 (red), and H-CuFeS2 (blue). Figure 10. Fenton-like reaction for RhB degradation in the presence of Na2S2O<sup>8</sup> by using different catalysts: Cu2S (black), FeS<sup>2</sup> (red), and H-CuFeS<sup>2</sup> (blue).	doab	2025-04-07T03:56:58.842254	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 123 }
004db1df-dc76-42af-952f-1f5e82b7822b.124	2.3. Degradation Mechanism of H-CuFeS<sup>2</sup> As a key mechanistic study, the active species involved in the degradation reaction were identified systematically using the free radical trapping experiments (Figure 11A). Methanol and NaN<sup>3</sup> were used as •OH and •SO<sup>4</sup> − scavengers, respectively. Comparing to methanol, NaN<sup>3</sup> inhibit RhB degradation more, indicating that •SO<sup>4</sup> − radicals are the major species involved in the Fenton-like degradation (blue curve in Figure 11A). According to the results of the scavenger test and XPS experiment, we propose a possible degradation mechanism. First, Fe2+/Cu<sup>+</sup> ions on the CuFeS<sup>2</sup> surface catalyzed S2O<sup>8</sup> <sup>2</sup><sup>−</sup> to produce •SO<sup>4</sup> <sup>−</sup> radicals (Equations (1) and (2)). Due to high oxidation activity of •SO<sup>4</sup> − radicals (E<sup>0</sup> = 2.5–3.1 V), they were utilized to degrade dyes and to oxidize Fe2+/Cu<sup>+</sup> ions (Equations (3)–(5)). Then, •OH radicals also produced from the oxidation reaction between •SO<sup>4</sup> <sup>−</sup> radicals and H2O/OH<sup>−</sup> to degrade the dyes (Equations (6)–(8)). Thus, after adding methanol to the reaction mixture, RhB degradation in CuFeS<sup>2</sup> samples was slightly decreased, indicating that production of •OH radicals are considered as the indirect active species in the CuFeS<sup>2</sup> catalyzed RhB degradation (red curve in Figure 11A). $$\text{Fe}^{2+} + \text{S}\_2\text{O}\_8\text{}^{2-} \rightarrow \text{Fe}^{3+} + \text{SO}\_4\text{-}^- + \text{SO}\_4\text{}^{2-} \tag{1}$$ $$\text{Cu}^+ + \text{S}\_2\text{O}\_8^{2-} \rightarrow \text{Cu}^{2+} + \text{SO}\_4\text{-}^- + \text{SO}\_4^{2-} \tag{2}$$ $$\text{CO}\_4\text{-}^- + \text{RhB} \rightarrow \text{CO}\_2 + \text{H}\_2\text{O} \tag{3}$$ $$\text{+SO}\_4\text{-}^- + \text{Fe}^{2+} \rightarrow \text{Fe}^{3+} + \text{SO}\_4{}^{2-} \tag{4}$$ $$\text{SO}\_4\text{-}^- + \text{Cu}^+ \rightarrow \text{Cu}^{2+} + \text{SO}\_4\text{2}^- \tag{5}$$ $$\text{SO}\_4\text{-}^- + \text{H}\_2\text{O} \rightarrow \text{SO}\_4^{2-} + \cdot\text{OH} + \text{H}^+ \tag{6}$$ $$\cdot\text{SO}\_4\cdot^- + \text{OH}^- \rightarrow \text{SO}\_4^{2-} + \cdot\text{OH} \tag{7}$$ $$\cdot\text{OH} + \text{RhB} \rightarrow \text{CO}\_2 + \text{H}\_2\text{O} \tag{8}$$ Figure 11. (A) Free radical trapping experiment and (B) absorbance spectra at different condition. Figure 11. (A) Free radical trapping experiment and (B) absorbance spectra at different condition. On the basis of the results described above, the degradation scheme of the H-CuFeS2 samples in the Fenton-like reaction was proposed (Scheme 1). •SO4− radicals and •OH radicals were produced from the Fenton-like reaction between S2O82− and Fe2+/Cu+ ions on the H-CuFeS2 surface to degrade RhB (Equations (1)–(8)). Then, Fe2+/Cu+ ions were regenerated through a series reduction of S2− anions (Equations (11)–(13)). Moreover, it is also possible to produce Fe2+ ions by reduction reaction between Cu+ and Fe3+ ions (Equation (14)). •SO<sup>4</sup> − radical production in the Fenton-like reaction was further studied using the spectrophotometric method [50]. According to Equations (9) and (10), I<sup>3</sup> − solution (light yellow) was found from chemical reaction between S2O<sup>8</sup> <sup>2</sup><sup>−</sup> and KI. The absorbance spectra of the S2O<sup>8</sup> <sup>2</sup>−/KI solution in the absence and presence of the prepared CuFeS<sup>2</sup> samples were evaluated. Figure 11B shows that an absorbance peak was observed at 358 nm for each sample and that the maximum absorbance was observed in the absence of the prepared CuFeS<sup>2</sup> samples (blue curve in Figure 11B). This suggests that S2O<sup>8</sup> <sup>2</sup><sup>−</sup> produced the highest amount of I<sup>2</sup> compared to others, thereby leading to more chemical reactions with KI to generate I<sup>3</sup> <sup>−</sup>. Due to a high specific surface area and high content of Fe2+ ions, H-CuFeS<sup>2</sup> effectively catalyzed S2O<sup>8</sup> <sup>2</sup><sup>−</sup> to produce •SO<sup>4</sup> − radicals, as a result of a few I<sup>2</sup> production. Thus, the absorbance intensity at 358 nm of H-CuFeS2/S2O<sup>8</sup> <sup>2</sup>−/KI mixing solution (black curve in Figure 11B) was lower than that of C-CuFeS2/S2O<sup>8</sup> <sup>2</sup>−/KI mixing solution (red curve in Figure 11B). $$\mathrm{S\_2O\_8}^{2-} + 2\mathrm{I}^- \to 2\mathrm{SO\_4}^{2-} + \mathrm{I\_2} \tag{9}$$ $$\text{I}\_2 + \text{KI} \rightarrow \text{I}\_3^- + \text{K}^+ \tag{10}$$ On the basis of the results described above, the degradation scheme of the H-CuFeS<sup>2</sup> samples in the Fenton-like reaction was proposed (Scheme 1). •SO<sup>4</sup> <sup>−</sup> radicals and •OH radicals were produced from the Fenton-like reaction between S2O<sup>8</sup> <sup>2</sup><sup>−</sup> and Fe2+/Cu<sup>+</sup> ions on the H-CuFeS<sup>2</sup> surface to degrade RhB (Equations (1)–(8)). Then, Fe2+/Cu<sup>+</sup> ions were regenerated through a series reduction of S2<sup>−</sup> anions (Equations (11)–(13)). Moreover, it is also possible to produce Fe2+ ions by reduction reaction between Cu<sup>+</sup> and Fe3+ ions (Equation (14)). Catalysts 2022, 11, x FOR PEER REVIEW 13 of 21 $$\rm{S^{2-}} + \rm{Fe^{3+}/Cu^{2+}} \rightarrow \rm{Fe^{2+}/Cu^{+}} + \rm{S\_2}^{2-} \tag{11}$$ $$\rm{^2S\_2^{2-}} + \rm{Fe^{3+}/Cu^{2+}} \rightarrow \rm{Fe^{2+}/Cu^{+}} + \rm{S\_n}^{2-} \tag{12}$$ $$\rm{S\_n}{^{2-}} + \rm{Fe}^{3+} / \rm{Cu}^{2+} \rightarrow \rm{Fe}^{2+} / \rm{Cu}^{+} + \rm{SO\_4}{^{2-}} \tag{13}$$ $$\rm Cu^{+} + Fe^{3+} \rightarrow Fe^{2+} + Cu^{2+} \tag{14}$$ Scheme 1. Possible scheme of Fenton-like reaction for the H-CuFeS2 samples. Scheme 1. Possible scheme of Fenton-like reaction for the H-CuFeS<sup>2</sup> samples. ### 2.4. Stability and Practical Applications of H-CuFeS2 2.4. Stability and Practical Applications of H-CuFeS<sup>2</sup> The stability of the catalyst is an essential parameter for the development of practical water treatment applications. To investigate the stability of H-CuFeS2, results of pH effect, copper ions effect, and cyclic RhB degradation tests were evaluated as shown in Figures 12–14. Figure 12 showed the study of pH effect. RhB degradation by H-CuFeS2 at pH 4.0 maintained a similar degradation efficiency at pH 7.0 (98.48% at pH 4.0 and 98.49% at pH 7.0, respectively), whereas that at pH 10.0 resulted in a considerable loss of efficiency (72.13% at pH 10.0). This is because most •SO4− radicals were converted to •OH radicals at alkaline condition (Equations (6)–(8)). Thus, •OH radicals are the major active radicals involved in dye degradation at alkaline condition. In addition, inactive porphyrin ferryl complexes (FeO2+) are formed as Fe2+ ions in the alkaline solution. As a result, a weakened The stability of the catalyst is an essential parameter for the development of practical water treatment applications. To investigate the stability of H-CuFeS2, results of pH effect, copper ions effect, and cyclic RhB degradation tests were evaluated as shown in Figures 12–14. Figure 12 showed the study of pH effect. RhB degradation by H-CuFeS<sup>2</sup> at pH 4.0 maintained a similar degradation efficiency at pH 7.0 (98.48% at pH 4.0 and 98.49% at pH 7.0, respectively), whereas that at pH 10.0 resulted in a considerable loss of efficiency (72.13% at pH 10.0). This is because most •SO<sup>4</sup> <sup>−</sup> radicals were converted to •OH radicals at alkaline condition (Equations (6)–(8)). Thus, •OH radicals are the major active radicals involved in dye degradation at alkaline condition. In addition, inactive porphyrin ferryl complexes (FeO2+) are formed as Fe2+ ions in the alkaline solution. As a result, a weakened degradation result at pH 10.0 was found (Figure 12). degradation result at pH 10.0 was found (Figure 12). In the study of copper ion effect as shown in Figure 13, RhB degradation efficiencies by H-CuFeS2 in the presence of Cu+ ioins were 88.64% at pH 4.0, 91.21% at pH 7.0, and 87.42% at pH 10.0, whereas those in the presence of Cu2+ ions were 93.96% at pH 4.0, 94.21% at pH 7.0, and 91.19% at pH 10.0. Comparing to that at pH 10.0 in the absence of copper ions, an obvious improvement was found. This is because •SO4− radicals are produced in the presence of Cu+ ions (Equation (2)). In addition, Fe2+/Cu+ ions were regenerated through reduction between S2− anions and Fe3+/Cu2+ ions (Equations (11)– In the study of copper ion effect as shown in Figure 13, RhB degradation efficiencies by H-CuFeS<sup>2</sup> in the presence of Cu<sup>+</sup> ioins were 88.64% at pH 4.0, 91.21% at pH 7.0, and 87.42% at pH 10.0, whereas those in the presence of Cu2+ ions were 93.96% at pH 4.0, 94.21% at pH 7.0, and 91.19% at pH 10.0. Comparing to that at pH 10.0 in the absence of copper ions, an obvious improvement was found. This is because •SO<sup>4</sup> − radicals are produced in the presence of Cu<sup>+</sup> ions (Equation (2)). In addition, Fe2+/Cu<sup>+</sup> ions were regenerated through reduction between S2<sup>−</sup> anions and Fe3+/Cu2+ ions (Equations (11)–(13)). As a result, an improve degradation at pH 10.0 was found. (13)). As a result, an improve degradation at pH 10.0 was found. system. Catalysts 2022, 11, x FOR PEER REVIEW 14 of 21 Figure 12. Fenton-like reaction for RhB degradation by the H-CuFeS2 samples at different pH value Figure 12. Fenton-like reaction for RhB degradation by the H-CuFeS<sup>2</sup> samples at different pH value system. system. Figure 12. Fenton-like reaction for RhB degradation by the H-CuFeS2 samples at different pH value Figure 13. RhB degradation efficiency by the H-CuFeS2 samples at different pH value system in the presence of copper ions. Figure 13. RhB degradation efficiency by the H-CuFeS<sup>2</sup> samples at different pH value system in the presence of copper ions. Figure 13. RhB degradation efficiency by the H-CuFeS2 samples at different pH value system in the the phase structure of the H-CuFeS2 samples after the repeated reactions, indicating the For recyling-used study, Figure 14A showed RhB degradation by H-CuFeS2 exhibited a considerable loss of efficiency (from 98.48% to 72.46% after three cycles). Furthermore, For recyling-used study, Figure 14A showed RhB degradation by H-CuFeS2 exhibited a considerable loss of efficiency (from 98.48% to 72.46% after three cycles). Furthermore, the corresponding XRD, Raman, and SEM results (Figure 14B–D) suggest a decrease in presence of copper ions. underway in our laboratory. destruction of the H-CufeS2 sample crystalization. In addition, EDS spectrum found that the atomic ratio (Cu:Fe:S) for the third used H-CuFeS2 samples was determined to be 1:1:1.9. The morphology of the third used samples still retained sphere-like structures, with the average diameter ranging 20–35 nm. Further research to improve recycling-used ability by other heterojunction, such as those doped by Ag@Ag3PO4 nanoparticles, is now Figure 14. (A) Fenton-like reaction for RhB degradation by the H-CuFeS2 samples for the recycling -used test, (B) XRD, (C) Raman spectra, and (D) SEM image of the 3rd used samples. Figure 14. (A) Fenton-like reaction for RhB degradation by the H-CuFeS<sup>2</sup> samples for the recycling -used test, (B) XRD, (C) Raman spectra, and (D) SEM image of the 3rd used samples. To assess the practical applications of H-CuFeS2 as a new water treatment option, various dyes (R6G, MB, and MO) and colorless organic compound (BPA) were tested (Figure 15A). H-CuFeS2 exhibited excellent degradation efficiency toward R6G, MB, MO, and BPA, with 96.84%, 93.86%, 81.89%, and 75.24% degradation achieved within 10 min, respectively. In addition, the mineralization performance of H-CuFeS2 comparing to a traditional Fenton reaction (Fe2+/H2O2) was evaluated. From the TOC analysis (Figure 15B), mineralization efficiency for the Fe2+/H2O2 and H-CuFeS2/S2O82− system was 70.0% and 80.1%, respectively, representing 10.1% improvement of RhB degradation. Finally, the prepared H-CuFeS2 samples were used to degrade RhB in the environmental water samples (pond water and seawater). H-CuFeS2 exhibited adequate mineralization effi-For recyling-used study, Figure 14A showed RhB degradation by H-CuFeS<sup>2</sup> exhibited a considerable loss of efficiency (from 98.48% to 72.46% after three cycles). Furthermore, the corresponding XRD, Raman, and SEM results (Figure 14B–D) suggest a decrease in the phase structure of the H-CuFeS<sup>2</sup> samples after the repeated reactions, indicating the destruction of the H-CufeS<sup>2</sup> sample crystalization. In addition, EDS spectrum found that the atomic ratio (Cu:Fe:S) for the third used H-CuFeS<sup>2</sup> samples was determined to be 1:1:1.9. The morphology of the third used samples still retained sphere-like structures, with the average diameter ranging 20–35 nm. Further research to improve recycling-used ability by other heterojunction, such as those doped by Ag@Ag3PO<sup>4</sup> nanoparticles, is now underway in our laboratory. ciency through the Fenton-like reaction for RhB degradation. A notable difference in the mineralization efficiency for RhB was observed for the seawater samples (47.9% efficiency within 10 min) compared with pond water samples (63.8% efficiency within 10 min), probably because of the effect of higher concentration of anions or radical scavengers in the seawater sample that reduced the degradation activity of H-CuFeS2. Nevertheless, the studies on the environmental water samples strongly support the benefits of this newly developed H-CuFeS2-based Fenton-like water treatment option. To assess the practical applications of H-CuFeS<sup>2</sup> as a new water treatment option, various dyes (R6G, MB, and MO) and colorless organic compound (BPA) were tested (Figure 15A). H-CuFeS<sup>2</sup> exhibited excellent degradation efficiency toward R6G, MB, MO, and BPA, with 96.84%, 93.86%, 81.89%, and 75.24% degradation achieved within 10 min, respectively. In addition, the mineralization performance of H-CuFeS<sup>2</sup> comparing to a traditional Fenton reaction (Fe2+/H2O2) was evaluated. From the TOC analysis (Figure 15B), mineralization efficiency for the Fe2+/H2O<sup>2</sup> and H-CuFeS2/S2O<sup>8</sup> <sup>2</sup><sup>−</sup> system was 70.0% and 80.1%, respectively, representing 10.1% improvement of RhB degradation. Finally, the prepared H-CuFeS<sup>2</sup> samples were used to degrade RhB in the environmental water samples (pond water and seawater). H-CuFeS<sup>2</sup> exhibited adequate mineralization efficiency through the Fenton-like reaction for RhB degradation. A notable difference in the mineralization efficiency for RhB was observed for the seawater samples (47.9% efficiency within 10 min) compared with pond water samples (63.8% efficiency within 10 min), probably because of the effect of higher concentration of anions or radical scavengers in the seawater sample that reduced the degradation activity of H-CuFeS2. Nevertheless, the studies on the environmental water samples strongly support the benefits of this newly developed H-CuFeS2-based Fenton-like water treatment option. Figure 15. Fenton-like reaction of (A) different dyestuff by the H-CuFeS2 samples, (B) TOC analysis of different degradation systems by using Fe(II) and the H-CuFeS2 samples in the different environmental water samples. Figure 15. Fenton-like reaction of (A) different dyestuff by the H-CuFeS<sup>2</sup> samples, (B) TOC analysis of different degradation systems by using Fe(II) and the H-CuFeS<sup>2</sup> samples in the different environmental water samples.	doab	2025-04-07T03:56:58.842987	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 124 }
004db1df-dc76-42af-952f-1f5e82b7822b.125	3. Materials and Methods ### 3. Materials and Methods 3.1. Preparation of CuFeS<sup>2</sup> 3.1. Preparation of CuFeS2 All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and were of analytical grade and used without further purification. In this study, hydrothermal (H) and co-precipitated method (C) were used to prepare CuFeS2 samples, representing as H-CuFeS2 and C-CuFeS2, respectively. For hydrothermal procedure, 0.989 g of CuCl, and 2.703 g of FeCl3·6H2O were added to 57 mL of deionized water, with stirring for 10 min. Then, 8 mL of Na2S ·9H2O (0.02 mol) was added dropwisely into the above green mixture. All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and were of analytical grade and used without further purification. In this study, hydrothermal (H) and co-precipitated method (C) were used to prepare CuFeS<sup>2</sup> samples, representing as H-CuFeS<sup>2</sup> and C-CuFeS2, respectively. For hydrothermal procedure, 0.989 g of CuCl, and 2.703 g of FeCl3·6H2O were added to 57 mL of deionized water, with stirring for 10 min. Then, 8 mL of Na2S ·9H2O (0.02 mol) was added dropwisely into the above green mixture. After stirring for 30 min, the black mixture was transferred into a Teflon-lined stainless-steel autoclave. The autoclave was sealed and heated in an electric oven at 200 ◦C for 10 h. After the autoclave naturally cooled to room temperature, the precipitates were centrifuged (5000 rpm, 15 min) and washed three times with ethanol and deionized water, and then dried in vacuum at 60 ◦C overnight. In addition, Cu2S and FeS<sup>2</sup> nanoparticles were prepared following similar method without adding FeCl3·6H2O and CuCl precursor, respectively. For the co-precipitated method, 4.95 mg of CuCl, and 0.0135 g of FeCl3·6H2O were added to 20 mL of deionized water, with stirring at 70 ◦C for 10 min. Then, 1 mL of NH4OH (30%) and 1 mL of N2H4·H2O (64–65%) were added dropwise into the above mixture with stirring at 70 ◦C for 3 h. After that, 0.024 g of Na2S·9H2O was added into the above brown mixture with stirring at 70 ◦C for 3 h. Finally, the black precipitates were centrifuged (5000 rpm, 15 min) and washed three times with ethanol and deionized water, and then dried in vacuum at 60 ◦C overnight.	doab	2025-04-07T03:56:58.843751	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 125 }
004db1df-dc76-42af-952f-1f5e82b7822b.126	3.2. Characterization of CuFeS<sup>2</sup> The morphological and compositional characteristics of all as-prepared samples were observed with scanning electron microscopy (SEM) on a HITACHI S-4300 (Hitachi, Tokyo, Japan) and transmission electron microscopy (TEM) on a 1200EX II (JEOL, Tokyo, Japan) equipped with a QUANTAX Annular XFlash QUAD FQ5060 (Bruker Nano, Berlin, Germany). The crystallographic texture of the samples was measured by powder X-ray diffraction (XRD) on SMART APEX II (Bruker AXS, Billerica, MA, USA) using Cu Kα radiation (λ = 1.5406 Å). Raman spectra were collected at room temperature using a confocal micro-Raman system (Thermo Scientific Inc., New York, NY, USA). A 532 nm laser line was used as the photoexcitation source with a laser power of 2 mW focused on the sample for 10 s. The binding energy of elements was determined through X-ray photoelectron spectroscopy (XPS) on a VG ESCA210 (VG Scientific, West Sussex, UK).	doab	2025-04-07T03:56:58.843888	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 126 }
004db1df-dc76-42af-952f-1f5e82b7822b.127	3.3. Degradation Procedure RhB degradation was used to assess the degradation activity of the prepared samples. For the Fenton-like reaction, 20 mg of the prepared catalyst samples was added into the RhB solution (20 ppm, 50 mL), and the solution was stirred in the dark for 30 min. At 10 min before adding Na2S2O8, the absorbance at its characteristic absorption peak of 550 nm was measured to check the adsorption ability of the prepared samples. Subsequently, 100 µL of Na2S2O<sup>8</sup> (2 M) was added to dye solution. After a given time interval, 1 mL of suspension was sampled with a plastic pipette and this aliquot was quenched immediately by adding 10 µL NaN<sup>3</sup> (1 M) and filtered by a 0.22-µm syringe filter organic membrane to remove catalyst particles. The concentration of RhB was measured using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments, Winooski, VT, USA) at its characteristic absorption peak of 550 nm. Similar processes were performed for other catalysts (Cu2S and FeS2), dyestuffs (R6G, MB, and MO), and organic pollutant (BPA). After the experiment, TOC concentration was determined on an Elementar Acquray TOC analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany) to evaluate the extent of mineralization.	doab	2025-04-07T03:56:58.843980	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 127 }
004db1df-dc76-42af-952f-1f5e82b7822b.128	3.4. Free Radical Trapping Experiment To investigate the active species generated during RhB degradation over H-CuFeS2, the trapping experiment was conducted using NaN<sup>3</sup> and methanol (each 0.1 M) as the capturing agent for •SO<sup>4</sup> <sup>−</sup> radicals and •OH radicals, respectively. The implemented trapping experimental procedure was identical to the steps mentioned in the degradation section with an additional step of adding the capturing agent at each run.	doab	2025-04-07T03:56:58.844085	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 128 }
004db1df-dc76-42af-952f-1f5e82b7822b.129	4. Conclusions The prepared H-CuFeS<sup>2</sup> samples showed higher RhB degradation efficiency through the Fenton-like reaction than the prepared C-CuFeS2, FeS2, Cu2S nanoparticles, and previously reported samples (Table 1). This high enhancement in the degradation efficiency (98.8% RhB degradation within 10 min) was attributed to the prepared H-CuFeS<sup>2</sup> samples possessed smaller size and higher surface area. Based on the results of scavenger test and radicals' quantitation experiments, H-CuFeS<sup>2</sup> catalyzed Na2S2O<sup>8</sup> to produce •SO<sup>4</sup> − radicals and •OH radicals for the organics degradation. As we know, the three limiting factors to address prior to industrial application were viable methods of catalyst preparation, the catalyst durability and universality under operating conditions. The prepared H-CuFeS<sup>2</sup> samples possessed several attractive features. First, the prepared H-CuFeS<sup>2</sup> samples in the presence of Na2S2O<sup>8</sup> had 98.8% RhB degradation performance within 10 min. In addition, various organics (R6G, MB, MO, and BPA) with 75.24–96.84% degradation efficiency could be achieved. However, the repeated use of H-CuFeS<sup>2</sup> showed performance deterioration due to the change in the crystal phase of used H-CuFeS2. Further research on the high recycling-used ability of other heterojunction CuFeS<sup>2</sup> composites, such as those doped by Ag@Ag3PO<sup>4</sup> nanoparticles, is now underway in our laboratory. Finally, the prepared H-CuFeS<sup>2</sup> samples were used to degrade RhB with 10.1% mineralization improvement comparing to traditional Fenton reaction (Fe2+/H2O2). It is also easy to recover H-CuFeS<sup>2</sup> catalyst comparing to Fe2+ ions. In addition, H-CuFeS<sup>2</sup> catalyst deposited on a cellulosebased substrate is ongoing in our lab. The difficult separation and recycle of powder catalyst may result in high cost and secondary pollution, therefore, the powder form of catalyst greatly limited the commercial industrial application. More importantly, H-CuFeS<sup>2</sup> deposited on cellulose is very suitable for the dynamic-flow water treatment system. We will propose a new adsorption-degradation strategy for the pollutant removal in industrial level application in the future. Table 1. Comparison of degradation performance using the (photo-) Fenton-like reaction. In summary, this study discovered the hydrothermal synthesis of CuFeS<sup>2</sup> samples and successfully demonstrated the application of the Fenton-like reaction in the environmental water samples. The current findings can be used to the application of AOPs in wastewater treatment in the future. Author Contributions: Conceptualization, P.-Y.W. and Y.-W.L.; methodology, Y.-W.L.; software, Y.-W.L.; validation, P.-Y.W., T.-Y.L. and T.W.; formal analysis, P.-Y.W., T.-Y.L. and Y.-W.L.; investigation, P.-Y.W.; resources, Y.-W.L.; data curation, P.-Y.W. and T.-Y.L.; writing—original draft preparation, Y.-W.L.; writing—review and editing, T.W. and Y.-W.L.; visualization, Y.-W.L.; supervision, Y.-W.L.; project administration, Y.-W.L.; funding acquisition, Y.-W.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Ministry of Science and Technology of Taiwan under contract (MOST 110-2113-M-018-001). Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.844144	6-5-2022 13:29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "004db1df-dc76-42af-952f-1f5e82b7822b", "url": "https://mdpi.com/books/pdfview/book/5163", "author": "", "title": "Heterogeneous Catalysis and Advanced Oxidation Processes (AOP) for Environmental Protection (VOCs Oxidation, Air and Water Purification)", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036535661", "section_idx": 129 }
0051d55d-461b-414c-a51b-301e45ff4d0c.0	Health Promotion in Children and Adolescents through Sport and Physical Activities • Antonino Bianco	doab	2025-04-07T03:56:58.849345	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 0 }
0051d55d-461b-414c-a51b-301e45ff4d0c.1	Health Promotion in Children and Adolescents through Sport and Physical Activities 2nd Edition Antonino Bianco Edited by Printed Edition of the Special Issue Published in Journal of Functional Morphology and Kinesiology www.mdpi.com/journal/jfmk	doab	2025-04-07T03:56:58.849421	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 1 }
0051d55d-461b-414c-a51b-301e45ff4d0c.2	Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition	doab	2025-04-07T03:56:58.849467	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 2 }
0051d55d-461b-414c-a51b-301e45ff4d0c.3	Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition Editor Antonino Bianco MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editor Antonino Bianco Department of Psychology, Educational Science and Human Movement University of Palermo PALERMO Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Journal of Functional Morphology and Kinesiology (ISSN 2411-5142) (available at: www.mdpi.com/ journal/jfmk/special issues/adolescents sport 2). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number, Page Range. ISBN 978-3-0365-1197-9 (Hbk) ISBN 978-3-0365-1196-2 (PDF) © 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.	doab	2025-04-07T03:56:58.849493	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 3 }
0051d55d-461b-414c-a51b-301e45ff4d0c.5	About the Editor #### Antonino Bianco Antonino Bianco (41 years old) is living in Palermo, Italy. He grew up in Resuttano (CL), a little town located in the middle of Sicily. Prof. Bianco is married to Esamuela Pieretta Mancuso and the Father of Barbara Alison Bianco. He graduated from Palermo University in Sport and Exercise Sciences; afterwards, he received a Ph.D. in Exercise Physiology (University of Palermo Medical School) and a Post-Doc in Exercise Physiology from Greenwich University (UK). He has worked for Palermo University since December 2008, and he has been an Associate Professor at the University of Palermo since November 2019. He has co-authored more than 150 peer-reviewed articles, including the GSSI Sports Nutrition Award at ECSS 2017. Of interest is that, at the age of 39, he was included within the list of the top Italian scientists in Sport and Exercise Sciences. His main research interests include: pediatric exercise and cognitive functions development; fundamentals of training methodology for muscle hypertrophy.	doab	2025-04-07T03:56:58.849600	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 5 }
0051d55d-461b-414c-a51b-301e45ff4d0c.6	Preface to "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition" The second edition of the Special Issue entitled "Health Promotion in Children and Adolescents through Sport and Physical Activities"has been successfully completed, as expected. As stated in the preface to the first edition, this Special Issue (SI) was initially intended to address a challenge in this field, but this over time topic is becoming an important cornerstone for scientists who are exploring the fascinating subject of pediatric exercise. I'm grateful to all contributors for choosing MPDI and in particular my Special Issue. > Antonino Bianco Editor ## Editorial Preface to "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition" Antonino Bianco Sport and Exercise Sciences Research Unit, University of Palermo, Via Giovanni Pascoli, 6, 90144 Palermo, Italy; [email protected] The second edition of the Special Issue entitled "Health Promotion in Children and Adolescents through Sport and Physical Activities" has been successfully completed, as expected. As stated in the preface to the first edition, this special issue (SI) was initially intended to address a challenge in this field, but this topic is becoming, over time, an important cornerstone for scientists who are exploring the fascinating subject of pediatric exercise. We open the second edition of this Special Issue with an interesting study protocol described by Ng et al., from the Health Research Institute, University of Limerick. The manuscript presents a useful overview of initiatives designed to encourage people to become more active and increase their awareness of the potential benefits of physical activity (PA). Chulvi-Medrano et al. present a 47-year comparison of lower body muscular power in Spanish boys. The authors conclude that a decline in lower body muscular power occurs in 10–11-year-old Spanish boys, which may be due to the increasing prevalence of sedentary lifestyles across Europe, particularly in the southern regions. Josip Karuc and Marjeta Mišigoj-Durakovi´c investigate the relationship between functional movement (FM) patterns, PA level, and weight status in an average adolescent population. As expected, the authors reinforce the consensus that overweight and obese adolescents exhibit poorer functional movement than normal-weight adolescents. Nemanja Lakicevic, a PhD student from Palermo University, presents evidence of the effects of alcohol consumption on recovery following resistance exercise. The systematic review was publicized on social media and provides an interesting overview of the potentially negative effects of alcohol, particularly in regard to adolescents. The acute cardiometabolic responses to multi-modal integrative neuromuscular training are reported by a pioneer in the science of modern pediatric exercise. Prof. Avery Faigenbaum is currently regarded as a major contributor in the field, often proposing new ideas and highlighting resistance training as the driving force behind the proper growth and development of coming generations. In line with Faigenbaum et al., Migliano et al. significantly contribute to this SI with their validation of cardiorespiratory fitness measurements in adolescents living in Texas (USA). Mustafa Sö ˘güt et al. report interesting data regarding anthropometric obesity indices, body fat percentage, and grip strength in young adults. Of interest, Roscoe et al. investigate accelerometer-based physical activity levels in British preschoolers. On the other hand, joint mobility among young people participating in free climbing has been investigated by Gasbarro et al. The ESA Program is also discussed in this Special Issue, in the contribution of Ewan Thomas et al., who consider the entire consortium involved in this innovative and sustainable European project (more than 60 people involved). The randomized controlled trial carried out by Chua et al. shows that four minutes of sprint interval training has no acute positive effects on alertness, mood, and memory in children. The study also provides useful information for physical education teachers interested in introducing new settings and scenarios to their lessons. The closing paper of the Special Issue is that by Tamara Rial and colleagues. Their investigation considers urinary incontinence (UI) among adolescent female athletes. Interestingly, the prevalence of UI among such athletes participating in Citation: Bianco, A. Preface to "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition". J. Funct. Morphol. Kinesiol. 2021, 6, 23. https://doi.org/10.3390/jfmk6010023 Received: 23 February 2021 Accepted: 24 February 2021 Published: 1 March 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). impact sports was most dominant and higher in those engaged in trampolining, followed by rope skipping. In conclusion, I wish to thank the MDPI Editorial Office, and particularly Molly Lu, for supporting me in the role of SI editor. I am also grateful to the EIC Giuseppe Musumeci for the valuable guidance he provided following several rejections. This Special Issue presents a total of 12 papers, encompassing 31 different affiliations, with authors from 12 different countries spanning three different regions of the world (Europe, North America, and Asia). I hope to continue the success of this Special Issue with a third edition in the near future. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.849694	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 6 }
0051d55d-461b-414c-a51b-301e45ff4d0c.7	Protocol Feasibility Study of the Secondary Level Active School Flag Programme: Study Protocol *Kwok W Ng 1,2,\ , Fiona McHale 1, Karen Cotter 3, Donal O'Shea <sup>4</sup> and Catherine Woods <sup>1</sup> Received: 27 February 2019; Accepted: 19 March 2019; Published: 26 March 2019 Abstract: Taking part in regular physical activity (PA) is important for young adolescents to maintain physical, social and mental health. Schools are vibrant settings for health promotion and the complexity of driving a whole-school approach to PA has not been tested in the Irish school context. The feasibility of the pilot programme of the Department of Education and Skills second level Active School Flag (SLASF) is needed. SLASF is a two year process that consists of the Active School Flag (ASF) certificate programme (year 1) and the ASF flag programme (year 2). This protocol paper is specific to the first year certificate process. Three schools around Ireland were recruited as pilot schools to carry out the year-long SLASF programme with 17 planned actions involving the entire school. Students in the transition year programme have a particular role in the promotion of PA in SLASF. Data collection consists of physical measures, accelerometers, survey data and interviews at the beginning and the end of the academic year. The primary focus on the feasibility of the programme is through process evaluation tools and fidelity checks consisting of implementation of the SLASF programme through whole-school surveys, focus group discussions of key stakeholder groups, as well as one-to-one interviews with a member of management at each school and the SLASF coordinator of the school. Secondary outcomes include PA levels and its social cognitive theories based correlates through physical health measures, surveys carried out pre- and post-intervention, as well as focus group discussions of the students. The results of this study are needed to improve the development of the SLASF through a predetermined stopping criteria and inclusion into systems thinking approaches such as the Healthy Ireland Demonstration Project. Keywords: physical activity; adolescent; health promotion; activePal; intervention #### 1. Introduction** There are multiple reasons—physical, psychological, social, environmental—health for adolescents to take part in regular physical activity (PA). However, adolescence forms a highly volatile stage in life where transitional periods can influence behaviour [1] and is a critical time for PA participation where habits—good or bad—developed, later persist into adulthood [2]. Despite the evidence of health benefits from PA, a clear reduction in PA levels is apparent alongside increasing age in adolescence. Data from the Children's Sport Participation and Physical Activity (CSPPA) study highlighted a decline in meeting the PA guidelines, at least 60 min of moderate-to-vigorous (MVPA) per day, from 18% among 12y olds (first year of second level education in Ireland) to 6% among 18y olds (last year of second level education in Ireland) [3]. Similarly, a systematic review and pooled analysis indicated that PA levels decline by a mean of 7% per year over this period [4,5]. By the age of 15y, on average, across 42 countries in the Health Behaviour in School-aged Children study, only 11% of girls and 21% of boys self-reported sufficient MVPA levels [6]. Global PA decline throughout adolescence, across a range of measurement units, appears as high as 60–70% [4]. Action is needed to reduce the drop in PA levels. Actions that target the drop in PA levels can include interventions at the individual, community and within policy [7]. Although policy interventions may provide the best return on investment [8], creating such changes requires a strong evidence base that interventions targeting the behaviours actually works. At the individual level, interventions that were designed to increase school-based PA levels have yet to demonstrate its effectiveness [8]. In particular, multi-component PA interventions have yet to demonstrate improvements in overall MVPA [9]. However, at the community level, school-based PA programmes have demonstrated some positive effect on overall PA levels [10]. #### 1.1. Whole-School Approaches to Physical Activity Promotion Successfully run school-based PA programmes includes quality physical education, physical activities before-, during- and after-school, at the school grounds, as well as activities based in the local community [11]. Haapala and colleagues [11] suggested that recess time activities before, during and after school are opportune moments to promote PA. Similarly, a study based on a 12 week walking intervention among girls who took part before-school and during recess times increased the time in light intensity PA by 10 min but differences in MVPA were not statistically significant when compared to the intervention group [12]. Some recent studies have investigated the role of peers in boosting PA levels. For example, peer leaders (15–16y olds) slightly older than the target group (13–15y olds) encouraged after-school activities. By the end of the 7-week after-school intervention, there was a statistically significant increase of three minutes of daily MVPA [13]. Same-aged peers were also effective in promoting six minutes of daily MVPA through diffusion messages among girls during the entire school day [14]. Statistically significant changes on PA levels were found for those identified as low active at baseline in an intervention that focused on increasing step count in both boys and girls [15]. Few school-based programmes managed to succeed in increasing boys' PA levels for school-based interventions through a peer-led model [16,17]. Programmes with in-class activities that promote PA by the class teachers are another way to increase PA levels [18]. The latter activities requires a whole-school approach towards PA promotion and in Ireland, this is known as the Active School Flag (ASF) [19]. The ASF (www.activeschoolflag.ie) is a Department of Education and Skills (DES) initiative supported by Healthy Ireland. It takes a 'whole-school approach' and requires all members of the school community to work together to strengthen the delivery of the physical education programme and to promote PA throughout the school in a fun and inclusive manner. It emphasises quality PE, co-curricular PA as well as partnerships with students, parents and the wider community. In 2018, 29% (N = 1329) of primary schools in Ireland had achieved ASF status. Some have the need for renewal, thus at the time of print, 722 primary schools currently have possession of an Active Flag. The journey towards achieving this status begins with self-evaluation in the areas of (i) physical education (ii) PA and (iii) partnerships. Following this, schools are expected to devise a strategic plan and implement changes to help improve the quality of PE, provide additional opportunities for PA and support additional involvement with the wider community. Lastly, the school has to provide a week-long focus on PA in an Active School Week. The ASF is well-established as a primary school initiative but has yet to be developed for the secondary level education sector. A generic model has been tried among secondary level schools, although it did not achieve the same effect because the uptake of secondary level schools is below 5%. The most notable differences were the lack of whole-school engagement in the secondary level schools. Feedback from those experiences suggested that the generic ASF in secondary level was viewed as a physical education programme as opposed to a whole-school initiative. Furthermore, there are structural differences between primary and secondary level schools, thus a direct translation of the primary ASF to the secondary level setting would be specious. Some examples of differences include; in primary schools, a single teacher generally remains with the same class throughout the entire day, whereas at secondary level schools, students are taught by multiple teachers. In the secondary level schools, many schools do not have a compulsory timetabled physical education in accordance with DES recommendations (i.e., a double timetabled period for all students every week). Secondary level schools tend to be larger and cater for more students than primary schools, yet facilities for promoting PA can be limiting. Peer influences tend to be stronger at secondary level education [20] and students will often choose to socialise with their friends and be sedentary rather than take part in PA [21]. In primary schools, all teachers are encouraged to take part in continuous professional development (CPD) as part of their classroom responsibility in the areas of physical education and PA. Class teachers in secondary level schools tend to be specialists in their subject area and may not have the confidence or competence to create active breaks in the classroom [22]. In the majority of Irish secondary level schools, there is the 'Transition Year' (TY) period, whereby it is non- examinable and students may go on to try out activities beyond the normal school curriculum [23]. The aims of TY are to "increase social awareness and social competence with 'education through experience of adult and working life' as a basis for personal development and maturity" [24]. TY programmes can cover various aspects of personal development, including visits to hospitals to learn about health behaviours [25], as well as taking part in other science programmes [26]. Many TY based programmes seek to develop youth leadership skills (i.e., Gaisce the President's Award, Young Social Innovators, Gaelic Athletic Association (GAA) future leaders, etc.). #### 1.2. Theoretical Background to the Study The theoretical premise of this feasibility study is underpinned by social cognitive theories [27] These theories are the most cited for health behaviour change interventions [28]. In these theories, there is a triad relationship between personal and environmental factors with behaviours being explored through individual level factors such as self-efficacy, stages of readiness for behavioural change and school related autonomy. In self-efficacy theory [29] there are four sources; mastery accomplishments, vicarious experiences, verbal persuasion and physiology are strong predictors of behaviour. Novice learners, such as students in TY, tend to have low confidence to promote PA at the beginning of the year [30]. When schools express their interest to be an SLASF school, a resource pack (see methods section) would support these students to improve the self-efficacy (opportunities for mastery accomplishments), create timetabled meetings with the purpose to focus on the SLASF (opportunities for vicarious experiences) and regular contact with others who are also carrying out the SLASF (opportunities for verbal persuasions), the sources of self-efficacy may create a better suited environment for promoting PA. Being autonomous in making decisions is known to be directly related to intrinsic motivations towards a behaviour [31]. However, in the school context, students may feel their overall autonomy is restricted to the bounds of the school. As a result, some students may find the school environment something that they can thrive in, whereas others may feel the environment restricts individual choices. The autonomy supportive environment is as important as having autonomy among school-aged children because it can lead to greater levels of PA outside of the school context [32]. Factors that may influence the autonomy or autonomy support can include the perception of school academic performance and the way teachers and peers support academic performance [33], perceptions of ability to make decisions in the school [34] and feelings of belonging in the school [35]. Furthermore, it has been reported that individual well-being is associated with individual's sense of autonomy [36], therefore studies may need to consider the mediating factors of student well-being. The levels of readiness for taking part in regular PA would vary vastly among the students throughout the school. According to the transtheoretical model [37] individuals are at different stages of their intention for behavioural change. Progression between the five noted stages include pre-contemplation (not ready), contemplation (getting ready), preparation (ready), action and maintenance. According to the model, the factors that influence moving between these stages consist of biopsychosocial factors, including self-efficacy, own and socially supported decisions and going through the process of change [38]. Through awareness of the stages of change among the target population, there is a greater understanding of how to create targeted interventions for the promotion of PA. #### 1.3. Measures of Feasibility The SLASF programme is in its pilot phase as part of a larger systems based Healthy Ireland Demonstration Project. It is also a feasibility study, because we aim to investigate whether it is suitable in secondary level schools and if so, how to do that [39]. According to Bowen and colleagues [40], designing feasibility studies can have eight areas of focus; acceptability, demand, implementation, practicality, adaptation, integration, expansion and limited efficacy. Although the SLASF is a non-clinical study, the feasibility study is useful to provide an indicator for stopping, revisions or continuing for a scaling up a randomised control trial [41]. Moreover, the predefined rules need to be put in place prior to the analysis to prevent uncontrollable biases [42]. In Table 1, Bowen and colleagues' areas of foci for this feasibility study are broken down into the areas which this study researches and stopping criteria. Table 1. Areas of focus for feasibility studies with measures and stopping criteria. #### 1.4. Purpose of the Study The primary objective for this study is to see if the SLASF certificate is an acceptable programme in secondary level Irish schools. The secondary objectives are to see how feasible it is to operationalise the components needed for testing a year-long intervention. These include collecting accelerometer and physical health measures of students in the schools, completion of survey questions for pre- and post-intervention evaluation and investigate areas for improvement from a pilot study to scale-up intervention programme. #### 2. Methods and Design #### 2.1. Setting Eligible schools include secondary level schools in the Republic of Ireland that have not previously applied for the SLASF. Special educational needs schools or schools without a range of students from each year group are excluded. The feasibility study will be conducted in three secondary level schools, covering the demographics of a girls only school, a mixed school with designated 'Delivering Equality of Opportunity in Schools' (DEIS) status and a mixed mainstream school. A DEIS school is part of the Department of Education and Skill's action plan for educational inclusion to address disadvantaged education. #### 2.2. Recruitment #### 2.2.1. School Recruitment The SLASF programme is a comprehensive programme and employs a whole school approach. As an intervention programme, schools were invited to take part if they are not currently in the process of obtaining the Active School Flag. The normal process of the SLASF recruitment is for schools to make an application but this was suspended to allow time to develop the new model. Schools that were interested in carrying out the feasibility study contacted the Active Schools office and requested to pilot the second level programme. There were three schools that responded and accepted because they each have a strong well-being structure in place in their schools. This is a feasibility study with secondary outcomes on efficacy hence matched control schools (one girls only, one mixed and one mixed DEIS school) were recruited to take part in the survey component of the study. No reserve list was formed for this cohort but engagement of all three schools throughout the year to follow the feasibility and evaluate the SLASF process. #### 2.2.2. Student Recruitment In the participating schools, there are two levels of student engagement—basic and comprehensive. Although all students are exposed to the same SLASF programme, those engaged in the SLASF Basic are required to complete less data collection measures than those in the SLASF Comprehensive. The whole school is involved in the SLASF Basic and a random class from each year group is selected to take part in the SLASF Comprehensive. Classes are known as mixed ability tutor groups. Students obtain their consent to take part in the study. Even though there may be some students who do not obtain consent or would not like to withdraw from the comprehensive aspect of the study, there is no likely reason for them to influence the feasibility of the study. The SLASF initiative is structured to fit into the TY framework providing real and meaningful opportunities for student voice and youth leadership. A TY class would be nominated by the school as the SLASF TY class. This TY class attends classes in relation to SLASF tasks including youth leadership, mentoring and PA promotion. #### 2.3. Consent All students are given information letters for their parents. The information stated the purpose of the study as well as components of the study that would involve their child. Also, the letter invites the parents to speak to their children about the study and there are contact details to the research team to answer questions the parents may have. Because of population bias from active consent, passive consent was used for all students in the basic part of the programme. The school manages the returned forms of withdrawal. This is because it is part of the whole-school process and as a feasibility of programme, schools need to be aware of how to handle withdrawal from the programme. The online survey requests the students to give their assent (if under the age of 16) or consent (if over the age of 16). Students assigned to the comprehensive study, receive the same information sheets as the basic programme but in addition, information about the other measures that are included as part of the feasibility and process evaluation. Parents are asked to give opt in consent due to the nature of data collection (i.e., use of accelerometers). As part of Irish law, students over the age of 16 can give their own consent to take part in the study. All students were asked to opt in to be included in video recordings of interviews. #### 2.4. Allocation Strategy There was no allocation strategy used to select the schools. As a feasibility study, it was important to test the SLASF in various contexts. Three contexts were chosen; a DEIS school, a girls only school and a mixed school. All schools are considered to be large in size and this would test out the possible whole-school approach. #### 2.5. Active School Flag Intervention The SLASF feasibility model is co-designed by a SLASF steering group and staff, researchers of the University of Limerick and feedback from the three lead schools. The SLASF process is designed to be peer-led by a TY SLASF class, who will have the support of an SLASF coordinator, SLASF committee members, school staff and school management. The initiative challenges peers to find ways to encourage more students in their school to engage in school-based PA opportunities (year 1—SLASF certificate) and community-based PA opportunities (year 2—SLASF flag). During year 1 (Active School Flag certificate) the focus is on increasing participation in school-based PA opportunities. Year 2 (Active School Flag) is focused on community-based activities. Previously the SLASF was viewed as a physical education initiative. In order to generate greater whole-school engagement, the SLASF tasks are formatted to draw support for the SLASF TY team from school management and teachers across a variety of different subjects. The new format of the SLASF process complements two current key educational initiatives: The Well-Being Framework by the Irish National Council for Curriculum and Assessment, the School Self-Evaluation process by the Department of Education and Skills and a new initiative presently at draft stage: The Parent and Student Charter also by the Department of Education and Skills. Students working towards Gaisce the President's Award can use their SLASF work to fulfil their Community or Personal Skill challenge requirement. Another benefit of SLASF is that it will link schools with the current national Healthy Ireland PA programmes and national youth charity events including: The SLASF programme is a whole-school approach to increase PA opportunities and generate opportunities for student voice and youth leadership. Currently, there are two levels. The first level is a certificate. This is open to all secondary level schools. It can serve as a good link from primary schools to continue on the ethos of active schools and allow a school to consider whether to take the next step or not. Moreover, SLASF is a DES (Department of Education and Skills) initiative and it can only be awarded to schools that adhere to physical education timetable recommendations that is, a double timetabled period of physical education for all year groups. This provision is not in place in a large number of secondary level schools, thus heretofore excluding them from the SLASF process. The introduction of the certificate level, without eligibility criteria, opens the SLASF process to all interested post primary schools. If the SLASF certificate proves beneficial it may encourage them to revisit their timetable policy. Achievement of the flag is a whole school process, meaning that management, staff and students all play a role in the programme. There is a requirement for website updates and an online presence. In order to achieve the SLASF Certificate a number of tasks must be completed during year 1. These include: (1) a staff slideshow, (2) an SLASF team slideshow, (3) class time slideshow (4) SLASF training day, (5) an SLASF awareness week (towards the beginning of the year), (6) website showcase, (7) SLASF whole school questionnaire, (8) SLASF launch event, (9) SLASF action plan, (10) 'Did You Know?' campaign, (11) PA module as part of Social, personal and health education (SPHE) subject for junior cycle students, (12) Active School WALKWAY, (13) Community Mapping of extra curriculum activities, (14) Community Event, (15) Active School Week, (16) SLASF accreditation visit and (17) school PA space audits (Figure 1). Figure 1. Second Level Active School Flag (SLASF) Intervention and Research Components Timeline. All 17 activities are scheduled throughout the school year including a combination of staff and students as the main actors in this process. The SLASF TY class should take leadership guided and supported by the SLASF coordinator and committee on the programme. A key part of the process is that the SLASF coordinator has timetable provision allocated to two class periods per week to carry out work with the TY class. The committee includes staff representatives from the school, including one from management, an SLASF coordinator, two other staff who work on the well-being curriculum to include SPHE, Civic, Social, Political Education (CSPE) and Physical Education teachers, as well as four youth leaders. Structure of different actors in the process can be viewed in Figure 2. Figure 2. Actors of the Second Level Active school Flag. Schools wishing to work towards the second level of the SLASF process, the Active School Flag, must have completed the certificate level and be able to confirm that they timetable physical education in accordance with DES recommendations. At the student level, there are three levels of involvement. There is the SLASF team, which is comprised of the SLASF TY programme group. There are four SLASF youth leaders who represent the team at the school committee level. To be a youth leader, the SLASF team member needs to apply for the position through an application process that is evaluated by the staff members of the SLASF committee. The youth leaders end up representing the student voice at the SLASF committee and have the responsibility of presenting the SLASF action plan and the SLASF end of year review to the school Principal. The third student level is the SLASF class representatives. There are two in every class in the school and students can also apply for this position through an application form. The selection will be made by the class tutor in consultation with the SLASF coordinator. #### 2.5.1. TY Leader Role The TY leader role is responsible for planning, promoting and implementing the SLASF initiatives and events throughout the school year. Based on self-efficacy theory [29], TY leaders are a closer connection to the students in the schools than teachers, thus strengthen vicarious experiences. Moreover, social support and leadership from the TYs can reinforce the basic premise of proactive behaviour change [43]. The SLASF team can be identified by being given pins to wear on their uniform. Moreover, part of the time spent on SLASF activities can be used as part of a time bank for other volunteering programmes, such as, Gaisce the President's Award. The selected SLASF youth leaders will receive their own distinct pins to wear on the uniform. #### 2.5.2. Activities for Certification For schools to be successful in achieving the activities needed for certification, there is a yearly planner with guideline dates for task completion that the schools will use to keep on track. This also includes the accreditation visit. For example, the SLASF slideshows for the staff, team and youth leaders need to have been completed before the 2nd week of the school year. A designated training day takes place a week later. The purpose of this training day is to introduce the pilot schools to each other and the research team. The research team describes the whole year process evaluation and measurements taken throughout the year. There are co-design opportunities between the TY leaders and researchers to formulate the surveys used to collect data. The training day is not expected to run every year once feasibility is over. At this training day, two members of staff, the SLASF coordinator and another on the SLASF team take four student leaders to a training venue to learn about how to run the activities throughout the year. The website should also go live at the outset of the process. The website should encompass an easy to find link to the SLASF section of the site and there are the four core parts of the SLASF process; Physical Education; PA; Partnerships; and Active School Week. A school census questionnaire was developed to help the SLASF team to identify their action plan. Core questions about PA opportunities, physical education, involvement in extra-curriculum activities and barriers to PA were included in an online survey. For a week after the awareness week, the survey is available for completion. All responses are anonymous and completed confidentially. Class teachers supervise and help answer any technical questions related to the completion of the survey. The data is stored on a secure server that is only accessible to the researchers. However, the overall results for each variable would be computed and provided for the school to carry out their own descriptive analyses with the TY group. The TY class can then produce meaningful findings from the survey to show the school through the notice board and used for one of the planned actions. For the TOW event a free rope and a 2 1/2 h workshop will be provided for each school. This will enable TYs to coach and officiate TOW competitions and SLASF committee members that complete the course will receive a TOW Community Coach certificate upon completion. Each class in the school will be involved in this event with each having 3 TOW teams that compete against each other during tutor time or physical education class to decide what team will represent the class. Each class TOW team competes against the other classes in their year group during lunchtime to find the best TOW team for their year. Local role models can be invited to help launch the event by taking part in one of the TOW teams. This event should take place before the mid-autumn break. After the mid-autumn break, the schools would have access to their school's results from the school questionnaire. The SLASF teams are given a month to review the results and start to design an SLASF action plan. At least three action points need to be agreed upon by the SLASF team. The proposed SLASF actions should be presented to the Principal for agreement. The agreed actions are then implemented in the second half of the school year. Towards the end of the year, the three agreed actions will be reviewed by the SLASF team members and presented to the school Principal during the last two weeks of the school year. In addition, all students in the selected year group in the school will take part in a four weeks PA module delivered by the Social and Personal Health Education (SPHE) teachers. There would have to be a 'Did You Know?' campaign around the school that helps raise awareness about the benefits of PA for teenagers, in particular the positive impact that PA has upon focus, concentration and academic achievement. Another practical task for the SLASF team is to signpost an Active School WALKWAY. The walkway is a route that can be used by the students in the school during recess time or under teacher supervision for active learning activities, before/after tests or during free classes. SLASF, in partnership with Get Ireland Walking, designed Active School WALKWAY packs consisting of colourful outdoor all-weather sign post plaques which include orienteering symbols. One of the tasks that the SLASF TY class have to undertake is to map, measure and erect the walkway signposts to create a school walking route. A school WALKWAY Day where all classes get the opportunity to complete the walkway route with their teachers on a nominated school day needs to be agreed by school management. Then it is organised and promoted by the SLASF TY team. The organised walkway can be used as part of orienteering activities during timetabled physical education, as well as other school-based initiatives. As the year ends, the school prepares for the accreditation visit for the certificate. A follow up visit takes place during the acquisition of the flag year. Prior to this, the school needs to organise a community mapping exercise and community events which should help with the design of the Active School Week (ASW) programme. The main aims of the ASW are to promote PA in a fun and inclusive way, as well as raising awareness about the availability and variety of PA opportunities for teenagers and their families in their local community. Throughout this week the school provides many and varied opportunities for staff and students to become more physically active throughout the school day. #### 2.5.3. Expected Outcomes Tables and Measures According to their training resources, the programme aims to impact on a number of areas. However, to measure them all, multiple sources are required. A collection of survey instruments can be used to measure some of the outcomes, whereas some interviews can be used to evaluate other outcomes. In addition, the programme is year-long and a whole-school approach, hence site visits and checking on progress through logbook entries would be used to determine the processes carried out during the study. In Table 2, there is a list of the areas that SLASF aims to promote and some measures that can be used to test these outcomes. #### Table 2. Aligning outcomes with measures. #### 2.5.4. Questionnaires There are two types of online surveys carried out throughout the year. The basic survey is completed by the entire school. This survey is anonymous and the focus is on PA participation and barriers to school related physical activities. This survey is a compulsory part of the SLASF process. Administration of the survey is decided by the school, with the intention to cover the entire school. Ideally, a census sweep of the school takes place at the same time. However, there may be some technical issues that may prevent this from happening. For example, schools may have a limited number of computers accessing the internet at any one time (bandwidth limits), may have a limited number of units to complete the survey (lack of tablets or computers) or could not get all the school to take part at the same time (timetabling issues). The results of the survey will be given back to the school for the purpose to plan specific school-based interventions. Therefore, it is important that the mode of data collection, analysis and reporting can be completed quickly and easily. Failing all technical capabilities to collect from an online platform, extra resources would be dedicated to ensure double coding from pen and paper surveys. The second type of survey is a comprehensive survey, used for evaluating the feasibility of the study. The participants in this study input their user-ID so that the data can be linked from the beginning and end of the year long programme. Completion of the online survey takes place as one of the testing stations during the data collection visits. All the students have tablets or allocated to a school computer to complete the online survey. Details of the instruments are reported in Table 3. #### Table 3. Battery of questionnaires. #### 2.6. Process Evaluation #### 2.6.1. Logbook Activities Each school is given a logbook to record their activities. This is used as part of the accreditation process and is used by the researchers to evaluate the processes that the school used. The logbook is mainly used by the SLASF team and the SLASF committees. Every week, the TY students have the opportunity to complete a small section in the diary to record what took place. The diary is linked to the school year and the expected timescale for carrying out specific activities. There is also a chart for the SLASF team to complete by recording the agreed actions to be carried out by the SLASF team. The team need to record the date of the agreed action, a short title for the action, the person(s) responsible, date of the action completed and a check box. The TY team carries out a brief version of the System of Observing Play and Leisure Activity in Youth (SOPLAY) [52]. SOPLAY is a direct observation tool that is used by the TYs to assess PA levels within specific PA areas in the school. Due to resources, the full SOPLAY protocol had to be reduced down to three specific areas around the school. Furthermore, the TYs use tablets to video record the specific area and retrospectively carry out the observations. It is designed in this way because the technology is more readily available in schools than the time when SOPLAY was created by McKenzie and colleagues [52]. Through, observing the video recordings, the results can be verified so the validity of the results are stronger. Trained researchers with the SOPLAY counting system can verify the results from the TYs by matching the observation results. The videos can also be used as part of a TY class, where the students can get an understanding of ways to record the different intensities of PA. The SOPLAY exercise is carried out over six times throughout the school year. The assignment of the dates are researcher assigned days. The TYs are informed of the audit in the morning of the day the recording takes place. To reduce potential bias in the results, the TYs are reminded not to tell others that they are carrying out the observation. Observations take place twice during lunchtimes, one 10 min into the beginning and the second when there is 10 min left. Another activity recorded in the logbook is the SLASF committee meetings. The logbook provides space for six meetings throughout the year. The meeting minutes include the people in attendance, the areas of discussion and the actions that were agreed. There is also space in the logbook has space for a list of agreed action created by the TY class during their timetabled class time. To encourage compliance, there is room for information such as the agreed action, the person responsible and the date for completion. In addition, there is room in the logbook for the TY team and coordinator to note activities that take place in a specific week. For each week, tasks that are suggested, such as the slide show, presentation of the action plan and so forth are available for the TY and coordinator to help remind to be on track. #### 2.6.2. Whole-School Surveys There are three surveys to be carried out by all students in the school. The whole-school survey is part of the feasibility study and is carried out through an online survey platform. It is a mandatory action to be carried out by the school and is carried out during the first two months of the academic year. The school uses this information for creating and implementing three school specific action plans. Within this survey there are details of participation levels and barriers to taking part in physical education and extra curriculum activities. Both staff and students complete a second survey halfway through the process with items also related to process evaluation. Items will test implementation, fidelity and satisfaction of the tasks completed to date. The final whole-school survey has items related to process evaluation and is completed towards the end of the academic year but before the accreditation visit. The survey will also be held on an online survey platform. Due to the difficulties in getting whole-school engagement towards the end of the school year, the survey has pragmatic evaluation items whereby it can be completed on a mobile device such as a tablet or smart phone. #### 2.7. Sample Size There are three schools that are part of the feasibility study. Unlike a sample size calculation, a justification is made for feasibility studies [53]. In Table 4, information about the size of the school, the type and the number of participants expected to complete the comprehensive arm of the study is presented. According to the Department of Education and Skills school lists, School B is one of the largest secondary level in Ireland, with 1313 enrolled students. It is also a DEIS school. Approximately 10% of secondary level students attend a DEIS designated school. Moreover, there are known socioeconomic barriers towards PA [6], therefore it is necessary to carry out this feasibility in a DEIS school environment. School A is an all-girls school. Almost one in five schools in the country are all-girls' schools. There are many reports of girls having lower levels of PA than boys and therefore it is essential to include an all-girl's school. School C has a slightly fewer number of students than the national average of 999 students per school. Moreover, the ratio between girls and boys is slightly higher for girls (1:1.05), whereas the national average in mixed schools tends to have fewer girls than boys (1:0.87). Table 4. Sample size descriptions. #### 2.8. Data Analyses #### 2.8.1. Quantitative Data The data from the surveys are analysed through relevant statistical methods for the follow up data in this feasibility study. Compatible data between comprehensive and basic surveys can be used to determine the test-retest reliability of the items given that a smaller subsample of the entire school. As reliability is an important psychometric property for question items, this is carried out during the first phase of data collection. Students take part in the comprehensive study have their measures taken two times during the academic year. The first time takes place in autumn 2018 and the second takes place six months later during the spring 2019. Accelerometer data are transferred through the ActivPal software based on 15sec epoch. The standardised cut-offs for different types of motion; sleep, standing, light, moderate and vigorous PA are then compared at an individual level from pre- and post-test time points. Similarly, the height, weight and grip strength data is compared between the time points and used to control the differences in accelerometer data. Comprehensive survey data is also analysed with differences in PA and school related factors. Exploratory approaches include cross-sectional multivariate analyses of PA and school-related factors as independent variables and device-based PA and perceptions of PA opportunities as the dependent variables. Mixed models and multi-level regression analyses can be used on the data that has sufficient follow up data from the first time point. The multi-level approach takes into account between- and within- individual processes that explain variances in the outcome measures. Through this approach, it is possible to test the extent of PA (psychosocial variables) and school-related factors in relation to changes in PA levels and opportunities, at the same time to examine the individual versus the school factors that contribute to the outcome variables. The follow-up data adds another level of analysis that can test the changes through the intervention. It makes it possible to examine, for example, the changes in PA levels across the schools from the beginning and the end of the study, while also taking into account changes in the psychosocial variables included in this study. The interactions between the contexts can confirm behavioural change theories by examining the mediating and moderation mechanisms in PA levels. The majority of the statistical analysis would be carried out using IBM SPSS. #### 2.8.2. Qualitative Data The majority of the qualitative data comprises of focus group data. The way data is captured is a summary of individuals who collectively agree and discuss on the content [54]. Therefore, the first phase of analysis is to provide quantitative analysis of the subjects and the group types [55]. Focus groups can be useful to find a consensus on a phenomenon, as well as to engage with participants to discuss and share ideas that would otherwise be difficult to gather from one to one interviews [56]. In particular, the structural approach to children's group research can be used and transferred across to adolescents so that the students' voice to be heard [57]. Because the way a person in the focus group may consider a way to respond to the moderators' questions could differ from what other individuals may be thinking at the time, it is important to consider the way individuals respond, with whom and in what ways [55]. Transcriptions are matched with assistant moderator notes of verbal and non-verbal behaviours. The data from one-to-one interviews is more straightforward. A semi-structure interview guide is used to direct the respondent to focus on the research questions and is used for further probing into these questions if the respondent needs to explain something further. Interviewees data are also merged with intonation coding to help reinforce the importance of non-verbal behaviour. The double coding from the transcription across the different qualitative approaches creates a rich source of data. The combination of data is inserted into NVivo software for qualitative analysis. The metadata and types of data are used to create a rich data set. The data undergoes a thematic analysis as suggested by Lederman [58] by (1) identifying the big ideas, (2) creating units of data, (3) categorizing the units, (4) negotiating categories and (5) identifying themes and use of theory. The theories surrounding social-cognitive theories, including self-efficacy theory [29], self-determination theory [31] and competence motivation theory [50] are lens used in the final steps of the content analyses. The data are collected through follow up measures throughout the year. The researchers incorporate verification checking at the beginning of each session to place a point where the respondents can focus on. In particular, we are interested in the processes of the intervention, as well as the potential transformation in beliefs, thoughts and actions over the course of the year. These steps are useful for designing the results in a way that allows for multi-method approach to the overall research questions. #### 2.8.3. Mixed Methods Analyses Both quantitative and qualitative data can complement each other. We hope that the data that derives from both methods of inquiry can be partly explained through the literature to date and other types of data that is collected. To return to the points of evaluation of the feasibility study, there are various numbers of expected outcomes that the school is expected to achieve and they are measured directed through particular sources (Table 2). For example, the expected outcome of a broad physical education curriculum is measured through the whole school survey on participation of various physical education activities. The data taken from the beginning of the year gives insight to the types of activities that the students reported to have attended in the past 12 months. Through data collection across all year groups, the survey data can be used to determine how broad the physical education programme actually is. The post-test survey would give an indication of the extent of the physical education programme. However, reliance solely on this measure may be limited to the actual item that is included in the survey [59]. Therefore, combining the data from focus groups by the students and staff at the school can give more details about what was popular, who experienced the changes and the mechanisms in place to make the broader physical education opportunities. Therefore, the focus on the results are on the processes of creating the change, thus allowing further insight into the behavioural change techniques used to facilitate such changes. The SLASF log data contains both quantitative and qualitative data and can be analysed for the percent of completion towards the SLASF. Actions in relation to SLASF throughout the year form descriptive feasibility analyses. Differences in the PA audit across the year are analysed through descriptive statistics over time. In combination with the logbook of actions and the results of the PA audit more details about the feasibility of schools' actions from the TY class can be determined in relation to desired outcomes. #### 2.9. Availability of Data and Materials After completion of the study, data will be stored at the University of Limerick's Data archive without potential identifiers and request for data can be made through the study's principal investigator (Last author). All supplementary materials for the SLASF programme including the resource pack, template logbook and accompanying resources will be available at https://osf.io/frx6t/. #### 2.10. Ethics Approval and Consent to Participate The study follows the principles of the Declaration of Helsinki. The study protocol has been approved by the research ethics committee of the Faculty of Education and Health Sciences, University of Limerick (ref no. 2018/10/18\_EHS). Written informed consent will be sought from participating teachers, students and students' guardians. All participants have permission to withdraw from the study at any time and data deleted if collected. In cases of important protocol changes, requests from the ethical committee will be sought for. Trial Registration: https://osf.io/keubz/register/ 5771ca429ad5a1020de2872e; Registered 24th September 2018; Clinical Trial Registration: NCT03847831. #### 3. Discussion In this year-long feasibility study of the SLASF, a mixed-method approach is used to give recommendation to stop, revise or conduct a randomised control trial. The whole-school approach requires multiple stakeholders, primarily the students in secondary level schools, the TY students, the SLASF staff and its committee, as well as the management. The theories used in this paper are based on social cognitive theories and stages of change model [29,31,50]. Whole-school based interventions in the promotion of PA have been increasing [11,19] although the inception of the SLASF in the secondary level schools is more complicated than primary level schools. The diversity of foci at secondary level schools brings challenges towards a uniform and national programme. This is evident to date, whereby 29% of primary schools are ASF schools, whereas less than 5% of secondary level schools have this status. Therefore, a feasibility study is needed to test the readiness prior to national roll-out. The results from this study would be used to help inform the development of the SLASF and report the experiences of the schools in the feasibility study. Secondary outcomes from the measures carried out in the study may lead to improved understanding of the mechanisms of the promotion of PA. Moreover, the direct mapping of the stated goals of the SLASF with measures would provide evidence. Future iterations of the SLASF may include opt in by the students to take part in the SLASF TY programme, thus providing a mixture of students who are active and inactive. The challenges to this programme include the fidelity of the year-long programme. Schools are dynamic systems all with different characteristics based on the people who attend it. Challenging aspects could include issues arising from the coordination of the staff and pupils to carry out the tasks. There may be other activities that take place in the school, which reduce the efforts needed to run the programme or conversely, highly engagement that roles are dispersed more than previously planned. Monitoring of fidelity and carrying out process evaluations would help inform the way the programme is run. This feasibility study is novel in design in that it a whole-school approach to the promotion of physical activity among adolescents who are empowered to organize activities over the course of the year. School management also receive an incentive by striving towards the goal and recognition of an Active School Flag. Successful piloting of the SLASF can lead to upscaling to all secondary level schools around the Republic of Ireland due to the programme endorsement by the Department of Education and Skills. Testing of the programme can be part of large scale RCT that would fit under the Healthy Ireland Demonstration Project. Author Contributions: K.W.N. drafted the manuscript. All authors were responsible for writing part of the manuscript and critically revising the complete manuscript. F.M. contributed to the background and concept of the study. K.C. contributed to the design of the programme. D.O. contributed to the study design. C.W. is the principal investigator and contributed to the concept and design of the study. All authors approved the final manuscript. Funding: The feasibility study is funded by Mayo Education Centre, Healthy Ireland and St. Vincent's Foundation. Acknowledgments: K.W.N., F.M., D.O. and C.W. are researchers who have remained independent in the design of the SLASF programme. K.C. is a member of the ASF steering committee and designed the SLASF programme. K.C.'s involvement was to ensure all the aspects of this protocol are correct. K.C. has no involvement with carrying out the research, either in data collection or analyses. Conflicts of Interest: K.C. is employed by the Mayo Education Centre although K.C. is not involved in carrying out the research. All other authors declare that they have no competing interests. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).	doab	2025-04-07T03:56:58.850161	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 7 }
0051d55d-461b-414c-a51b-301e45ff4d0c.8	Brief Report A 47-Year Comparison of Lower Body Muscular Power in Spanish Boys: A Short Report *Iván Chulvi-Medrano 1,\ , Manuel Pombo 2, Miguel Ángel Saavedra-García 3, Tamara Rial Rebullido <sup>4</sup> and Avery D Faigenbaum <sup>5</sup> Received: 11 July 2020; Accepted: 18 August 2020; Published: 20 August 2020 Abstract:** Much of the evidence examining temporal trends in fitness among youth has found a decrease in measures of muscular strength and muscular power over recent decades. The aim of this study was to examine trends in lower body muscular power in Spanish boys over 47 years. In 1969 140 boys (10–11 years; body mass index = 19.24, SD = 2.91 kg/m2) and in 2016, 113 boys (10–11 years; body mass index = 19.20, SD = 3.15 kg/m2) were recruited. Lower body power was assessed using the vertical jump (VJ) and standing long jump (SLJ) tests. Significant differences and a large effect size were shown between groups in the SLJ (p = 0.001; d = 0.94) and the VJ (p = 0.001; d = 0.66). SLJ data in 1969 were higher (1.52 m, SD = 0.19) when compared to the 2016 data (1.34 m, SD = 0.18). The VJ performance of the 1969 sample was also higher (25.95 cm; SD = 6.58) than the 2016 sample (21.56 cm; SD = 4.72). SLJ and VJ performance of the 2016 group decreased 11.8% and 16.9%, respectively. There were no significant differences between groups in body mass index. The results indicate a secular decline in lower body muscular power in 10–11-year-old Spanish boys with no significant changes in body mass index over the 47-year study period. Keywords: pediatric dynapenia; children; resistance training #### 1. Introduction Low levels of muscular fitness (i.e., muscular strength, muscular power and local muscular endurance) in children and adolescents are associated with poor motor competence, functional limitations and adverse health outcomes [1,2]. Recent findings indicate that measures of muscular strength and power in modern-day youths are lower than in previous generations [3–6]. Sandercock and Cohen reported a decline in muscular fitness (bent-arm hang, sit-ups and handgrip) using allometric equations in 10-year-old English children from 1998 to 2014, and noted this trend was independent of secular changes in body size [5]. A similar trend in muscular fitness was observed in Spanish adolescents between 2001–2002 and 2006–2007 [4], and in an international sample of children and adolescents between 1964 and 2017 [7]. Lower levels of muscular strength and power in modern day youths appear to be consequent to lifestyles characterized by reduced physical activity and increased sedentary behavior [3,5,8]. Importantly, muscular strength and fundamental movement skill proficiency are considered foundational for ongoing participation in physical activity across the lifespan [8,9]. Therefore, it is critical to examine temporal trends in muscular fitness in youth due to the far-reaching implications for disease prevention and health promotion. A recent meta-analysis concluded that poor muscular fitness was associated with lower levels of bone mineral density and self-esteem, as well as higher levels of body fat and cardiometabolic risk [1]. In support of these observations, lower handgrip levels in youth with obesity have been associated with increased cardiometabolic risk [10]. Further, low levels of performance on selected measures of muscular fitness, including the handgrip, push-up and long jump early in life have been found to be associated with an increased risk of metabolic syndrome later in life [11]. Specifically, the long jump is a field test commonly used in youths as a general measure of lower body muscular fitness [12]. Temporal trends of muscular fitness performance in youth can be used to inform public health policies about youths' physical activity and health [13]. However, there are a limited number of studies examining trends in lower body muscular fitness in youths over recent decades. Additional data are needed to fill this research gap. The aim of this study was to describe temporal trends in lower body muscular power in Spanish boys over a 47-year time period from 1969 to 2016. We hypothesized that contemporary trends towards decreasing levels of muscular strength and muscular power shown in previous studies will be similar in Spanish children in 2016 as compared to Spanish children in 1969. #### 2. Materials and Methods Data were collected from two separate cross-sectional samples from the same school in Galicia, Spain. The sample consisted of the total boys (10 to 11 years of age) enrolled in the school during both years. Participants included 140 boys (10–11 years; body mass index = 19.24, SD = 2.91 kg/m2) in 1969 and 113 boys (10–11 years; body mass index = 19.20, SD = 3.15 kg/m2) in 2016. The University Da Coruña Ethics Committee approved this research study and parents and participants were informed about experimental procedures and provided parental permission and child assent, respectively. Body mass index (BMI) was calculated as body mass measured on an analogic scale to the nearest 0.1 kg divided by height measured on a stadiometer to the nearest 0.5 cm squared. Lower body power was assessed using the vertical jump (VJ) and standing long jump (SLJ) tests following standardized procedures [14]. In the SLJ the participant stood with both feet just behind the starting line on a marked floor. The distance between the starting line and the back edge of the participant's heel was measured after each jump. For the VJ test, participants were instructed to jump as high as possible and mark the wall with chalk on their fingers. The vertical jump was calculated by subtracting a participant's standing reach height from the maximal jump height. Participants were permitted to perform a countermovement prior to jumping vertically or horizontally. The test order was randomized, and all participants performed 3 trials per test. Participants were allowed to rest for 1 min between trials and for 3 min between the VJ and SLJ tests. The best score from each test was used for data analysis. In 2016, the same testing protocols and procedures were followed as in 1969. The VJ and SLJ were part of the Spanish physical education curricula during the study period and therefore all participants had 2 familiarization sessions with these tests. A Kolmogorov–Smirnov test was conducted to determine whether the data met the assumptions of normality of distribution. When the data met the normal distribution, two-tailed t-tests were applied for normally distributed data and the Mann–Whitney test was used for non-normal data to determine differences between cohorts. Significance was stablished at p < 0.05. D-Cohen effect size (ES) was calculated using the recommended Equation (1) [15]. $$\text{ES} = \frac{\text{mean of the experimental group -- mean of the control group}}{\text{standard deviation of the control group}} \tag{1}$$ #### 3. Results In 1969, the sample mean age was 10.53 (0.50) years-old (min 10- max 11-years-old) and the median was 11-years-old. In 2016, the sample mean age was 10.43 (0.49) years-old (min 10- max 11-years-old) and the median was 10-years-old. Characteristic outcomes in the different cohorts are presented in Table 1. Table 1. Participant demographics and lower body muscle power over a 47-year-period in Spanish boys. <sup>1</sup> BMI: Body Mass Index; SLJ: Standing Long Jump; VJ: Vertical Jump. #### 4. Discussion Significant differences and a large effect size were found in SLJ and VJ performance from 1969 to 2016. Over this 47-year period, SLJ performance decreased 11.8% and VJ decreased 16.9%. These results indicate a declining trend in lower body muscular power in 10–11-year-old Spanish boys with no significant changes in BMI. Our findings show a greater decline in SLJ than in previous research. We observed an 11.8% decline in SLJ between 1969 and 2016, whereas Moliner-Urdiales and colleagues found a decline in SLJ performance of 4.8% in 12.5- to 17.5-year-old Spanish adolescents assessed between 2001–2002 and 2006–2007 [4]. Similarly, Hardy and colleagues reported a decline in SLJ in youth between 1985 and 2015, with 10-year-old boys decreasing SLJ performance by 4.8% [13]. These trends in measures of lower body muscular strength and muscular power in youth may be explained by declining levels of regular participation in physical education, outdoor active play, and sport activities during the respective study periods [16]. Of interest, Tomkinson and colleagues quantified global changes in anaerobic fitness in more than 20 million youths and reported improvements in performance from 1958 to 1982, followed by a plateau and eventual decline in anaerobic test performance until 2003 [17]. This observed decline in anaerobic performance is consistent with our observed temporal trends in musculoskeletal fitness. Similarly, Kaster and colleagues evaluated temporal trends in sit-up performance in almost 10 million children and adolescents and reported large international improvements from 1964 to 2000 before then stabilizing near zero until 2010 before declining [7]. Of note, national trends in sit-up performance were strongly and positively associated with trends in vigorous physical activity, with countries with the largest improvements in sit-up performance reporting the largest increases in vigorous physical activity [7]. Another outcome of interest is the BMI. Our findings indicate that there was no significant difference in BMI between 1969 and 2016. However, the 2016 boys were shorter with a lower body mass than in 1969. These differences could be attributed to a younger age of development. While BMI in our study showed no significant differences over a 47-year period, other reports observed an increase in BMI over a period of 30 years [13]. Our findings are not consistent with others who reported that today's youths are taller and heavier than previous generations [13]. Of interest, the Spanish physical education curricula in the 1960s, 1970s and 1980s typically included more strength- and skill-building physical exercises, as compared to the more recent focus on aerobic games and activities. As such, trends in BMI and body composition must be viewed in light of the type and intensity of exercise performed, as well as the age, sex and biological maturation of the study participants. We found a decrease in lower body power independent of changes in body size and BMI over the study period. The same downward trend in muscular fitness independent of secular changes in body size and BMI was also observed in 10-year-old English children, as reported by Sandercock and Cohen [5]. While our 1969 sample was heavier and taller than our 2016 sample, with no significant differences in BMI, English children in the Sandercock and Cohen study were taller and heavier in 2014 than in 2008 and 1998, with no significant differences in BMI [5]. Collectively, these results suggest that changes in muscular fitness over time may not be associated with secular changes in BMI and body size. It is possible that other factors, such as trends in quality and quantity of physical activity engagement, may contribute to the observed decline in muscular fitness in modern day youths. This downward trend in muscular fitness in modern day youths has also been reported in children and adolescents from other countries [3]. It should be noted that, without regular opportunities to engage regularly in strength-building exercises, today's youths may be less likely to attain the adequate levels of muscular fitness that are needed for ongoing participation in MVPA [3,18]. Since low levels of muscular strength and power early in life are risk factors for pediatric dynapenia and associated health-related concerns [19], the SLJ has been suggested as a valid general index for assessing muscular fitness in youths [12]. Our results show that the 2016 cohort was shorter and their SLJ performance was significantly lower than in 1969. In young adults, SLJ performance has been found to be influenced by several factors, including anthropometrics [20]. Taller subjects have a higher center of gravity and longer leg lever that can produce greater mechanical jump forces. It has been reported that femur length has a significant influence on VJ performance [21]. In 10- to 12-year-old children, a positive moderate correlation between femur length and SJL, and a weak positive correlation between standing height and SLJ, were reported [22]. Given these observations, participant's anthropometrics may affect the take-off angle during the SLJ and, consequently, the jump distance. Our study has several limitations that should be considered when interpreting the data. Notably, we included a relatively small sample of boys from Galicia, a region of Spain. Therefore, our data are not representative of all Spanish youths. Additionally, we did not assess the biologic maturation of the participants and we did not measure their current levels of physical activity with validated questionnaires. Additionally, we did not include the periodic testing of muscular fitness during the 47-year study period and, therefore, changes in performance during selected periods of time cannot be analyzed. The assessment of body composition in order to differentiate between lean body mass and fat mass was not performed. Additional research is warranted to address the changes in muscular phenotype (e.g., muscular strength and power) in girls and boys, while controlling for confounding variables, such as exercise participation and training history. Finally, it has been reported that sociodemographic variables [13] and the trends towards unhealthy eating habits [23] may influence fitness performance in youths; however, we did not assess these variables. #### 5. Conclusions To the best of our knowledge no previous studies have examined temporal trends in muscular fitness in youths over a 47-year period. Our novel findings are consistent with other reports from western societies that highlight declines in measures of muscular strength and muscular power in modern day children and adolescents. To alter the current trajectory towards lower levels of muscular fitness in children and adolescents, developmentally appropriate interventions that target neuromuscular deficiencies and enhance muscular strength and power are needed to avert the troubling consequences of pediatric dynapenia. Author Contributions: Conceptualization, M.P. and I.C.-M.; methodology, M.P. and M.Á.S.-G.; software, M.Á.S.-G.; formal analysis, I.C.-M., M.Á.S.-G., T.R.R., and A.D.F.; data curation, M.Á.S.-G, I.C.-M., T.R.R., and A.D.F.; writing—original draft preparation, I.C.-M., M.P., and M.Á.S.-G.; writing—review and editing, T.R.R. and A.D.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).	doab	2025-04-07T03:56:58.854060	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 8 }
0051d55d-461b-414c-a51b-301e45ff4d0c.9	Review Relation between Weight Status, Physical activity, Maturation, and Functional Movement in Adolescence: An Overview #### *Josip Karuc \ and Marjeta Mišigoj-Durakovi´c Faculty of Kinesiology, University of Zagreb, Horva´canski zavoj 15, 10 000 Zagreb, Croatia; [email protected] \* Correspondence: [email protected]; Tel.: +3859-1582-3504 Received: 4 April 2019; Accepted: 29 May 2019; Published: 30 May 2019 Abstract: Obesity, low level of physical activity and dysfunctional movement patterns presents one of the leading health issues that can contribute to increased risk for developing not only metabolic and cardiovascular disease, but also musculoskeletal problems. The aim of this paper is to summarize literature and evidence about relationship between functional movement (FM) patterns, physical activity (PA) level and weight status in average adolescent population. In addition, this paper summarized current evidence about relations between maturation effects and functional movement among athletic adolescent populations. Summary of current evidence suggests that decreased physical activity level is negatively correlated to functional movement in adolescence. Additionally, most studies suggest that weight status is negatively correlated to functional movement patterns although there is conflicting evidence in this area. Evidence consistently showed that overweight and obese adolescents exhibit poorer functional movement compared to normal weight adolescents. In addition, it appears that maturation has effects on functional movement in athletic populations of adolescents. It is therefore important that practitioners consider interventions which develop optimal functional movement alongside physical activity and weight management strategies in children, in order to reduce the risks of injuries and pathological abnormality arising from suboptimal movement patterns in later life. Keywords: FMS; pubescence; pediatric population; fundamental movement #### 1. Introduction According to WHO, obesity has tripled since 1975 and thus represents one of the leading world health problems [1]. Along with the obesity and overweight, low level of physical activity (PA) puts overweight children at a higher risk for developing noncommunicable diseases. Although PA level is important for health of the locomotor system and represents a quantitative measure of human movement, due to importance of locomotor health, qualitative aspects of movement need to be considered as well. Functional movement (FM) considers qualitative aspects of movement, and can be defined as optimal postural control and mobility of joints and body regions involved in a particular movement. On the other hand, dysfunctional movement patterns present low level of quality of FM and can be related to injury incidence and thus endanger musculoskeletal (MSK) health and can contribute to developing degenerative changes in adulthood. Looking altogether, obesity, low level of PA, and dysfunctional movement, can contribute to even more increased risk for developing not only metabolic and cardiovascular disease, but also MSK problems in adulthood. Therefore, literature about mutual adverse effects of dysfunctional movement on mentioned variables need to be considered in order to provide practical information for professionals in the field of kinesiology, medicine, and related areas. The importance and influence of PA and weight status on adolescent health has been investigated widely, however only few studies have examined the relationship between FM, PA level, and obesity among the pediatric population. To date, only few studies have examined FM in the general adolescent population [2–11]. There are only four studies that investigated relation between weight status and FM in children [8–11]. Additionally, only one study by Duncan and Stanley, investigated the association between FM and PA level among children [10]. However, no study appears to have summarized literature about functional movement and its relation to PA level, weight status, and maturation in the average adolescent population. Therefore, the aim of this paper is to summarize literature and evidence about the relationship between FM patterns, PA level, and weight status in the average adolescent population. Additionally, this paper will summarize current evidence about relations between maturation effects and functional movement among the athletic adolescent population. #### 2. Materials and Methods The author of this study conducted a search in PUBMED (from 1 January 1990 to 1 May 2019) searching for association between functional movement, weight status, physical activity level, and maturation in adolescent populations. Key words used for electronic searches were: "functional movement", "functional movement screen", "weight status", "physical activity", "physical activity level", and "maturation". Combining the key words "functional movement" and "functional movement screen" with the other key words: "adolescents", "intervention", "weight status", "physical activity", "physical activity level", and "maturation" were used according to Boolean logic. The studies were checked by one researcher (J.K.) and were searched by title/abstract. In this study, inclusion criteria were (1) studies that investigated functional movement on average adolescent population and exercise intervention aimed to improve functional movement outcomes in average adolescents, (2) studies that examined the association between functional movement and weight status and physical activity level in average adolescent populations, (3) studies that investigated the association between functional movement and maturation in athletic adolescent populations, and (4) English as a publication language. Exclusion criteria in this study were (1) studies that investigated adult populations, special populations (e.g., firefighters, officers, military population etc.) and populations with specific diseases or injuries, and (2) studies that investigated functional movement skills (FMS) or functional capacity/competence in pediatric populations were excluded as well. In addition, manually selected papers from references within selected researches were included in this paper. #### 3. Results After an electronic search, the total number of studies that investigated functional movement via functional movement screen was 202. After inclusion and exclusion criteria were met, and after manually selection of references, 14 studies were included in this narrative review. After search, studies were categorized into three distinct areas: (1) functional movement in the average adolescent population and exercise intervention aimed to improve functional movement outcomes (six studies selected), (2) physical activity, weight status and functional movement among the adolescent population (four studies) and (3) maturation and functional movement among the athletic population of adolescents (four studies). Each of these study areas are incorporated and interpreted in the discussion section. #### 4. Discussion** #### 4.1. Clinical Importance of the Functional Movement and Functional Movement Screen as a Diagnostic Tool The Functional Movement ScreenTM (FMSTM), originally created by Gray Cook and Lee Burton, is a screening instrument intended to evaluate deficiencies in mobility and stability [12,13]. FMSTM includes seven tests: the deep squat, hurdle step, inline lunge, shoulder mobility, ASLR, trunk stability push-up and rotary stability. To the author's knowledge, this is the only diagnostic instrument available, described and validated in scientific literature for the purpose of screening functional movement patterns. Scoring the FMSTM has its own rules and procedures. The FMS raters use a standardized procedure to evaluate movement function. While performing FMS testing, each participant has a maximum of three trials for each test in accordance with the recommended protocol. Each test is scored on a three-point scale, from 0 to 3, with higher scores indicating better FM. In the presence of pain, a score of zero is noted. For each test, the highest score from three trials is recorded. An overall composite score was calculated with a total FMS score of 21 according to standardized guidelines. Descriptions of each FMS test, as well as standardized guidelines for the complete FMS testing is well written in the literature and can be studied elsewhere [12,13]. Some studies have shown the efficiency of the FMS in determining injury risk in athletes [14–16], however, others indicated the opposite [17–19]. Although there is conflicting evidence about the FMS as an injury predictive tool, the author's opinion is that the FMS has critical value for identifying movement mobility and stability deficiencies. Deficits in movement mobility and joint stability potentially predispose athletes and average populations to higher injury risk since optimal movement patterns can possibly prevent and reduce that risk. Several studies reported moderate to good inter-rater and intra-rater reliability of the FMS even among novice raters [20,21]. In addition, two-hour education on using FMS as a diagnostic tool seems to be efficient according to prior research [21]. #### 4.2. Functional Movement in the Average Adolescent Population and Exercise Intervention aimed to improve Functional Movement Outcomes Although there are a number of studies that investigated functional movement among athletic adolescents, only few studies investigated FM in the average adolescent population. These studies investigated relations between FM and PA or weight status. Additionally, few studies investigated maturation effect on functional movement in the athletic adolescent population. Only one study provided normative values for the FMS in the adolescent population [2]. Another study investigated the prevalence of functional movement patterns in children over the first three years of post-primary education [3]. Abraham et al. [2] investigated functional movement patterns in the average population of adolescents. This study included a large number of participants (n = 1005) with ages from 10 to 17 years old. They reported a mean value of the total FMS score of 14.5 points. Additionally, results showed a significant difference in total FMS score between females and males. However, no significant difference in scores existed between those who reported a previous injury and those who did not. In addition, the authors suggested normative values for the individual functional movement patterns for this population that can be found in their paper [2]. However, there are few limitations of this study that should be considered while implementing normative values in the practice. Large age span (10–17 years) among participants in this study reveals that pre-pubertal and pubertal subjects were included in the sample. Additionally, this study excluded all inactive children which could potentially lead to higher mean values. Although there are few limitations in this study, this is the first study that provided normative values for a school aged adolescent population. Lester et al. [3] examined the age-related association of functional movement among children in Ireland. The aim of this study was to gather data on prevalence of movement skills and functional movement patterns in children over the first three years of post-primary education (n = 181, mean age = 14.4). In this research, 43.6% of adolescents were in year one, 23.8% of adolescents were in year two and 32.6% adolescents were in year three with age range from 12.3 to 16.4 years old. Looking altogether, authors reported that results of the functional movement outcomes in their sample were suboptimal across all years. As authors stated, significant age-related differences were reported. When we look this data, as age increases, scores on the in-line lunge pattern decrease (difference between the first and third year). Additionally, the mean total FMS score reported in this study was 14.05, which is similar to results obtained by Abraham et al. The authors of this study strongly suggest that school-based intervention should be incorporated across the post-primary education child population in order to decrease decline in the impaired movement patterns. Four studies investigated the impact of exercise intervention on functional movement outcomes. Coker [4] investigated the impact of the standardized warm-up protocol in middle school children on functional movement parameters. Participants from seventh-grade and four eighth-grade physical education classes participated in this study (n = 120, mean age = 13.1 years old). A six-week intervention included exercises that targeted mobility and stability of joints and muscle activation (exercise targeted ankle joint mobility, pelvic stability and dysfunctional gluteal, abductors, and adductors muscles). Results of this study suggest that a warm-up, which consists of exercises that target typical movement and body dysfunctions among adolescents of a sensitive age, can significantly reduce dysfunctional movement patterns. It is the author's opinion that school policies should implement these programs into the physical education curriculum in order to reduce dysfunctional movement pattern prevalence and potentially reduce risk injury incidence among the average adolescent population. A study done by Nourse et al. [5] investigated the impact of live video diet and exercise intervention on vascular and functional outcomes in overweight and obese children (mean age = 14.5, n = 20). The intervention lasted 12 weeks and included three times per week videoconferences with a trainer and diet consultations. Results of this study showed a significant reduction in waist-hip ratio and improvement in total functional movement screen score. Average improvement of the participants in total FMS score was 13 to 17 points, from baseline to the end of this intervention, respectively. Authors of study concluded that a 12-week live video intervention improves functional movement outcomes in the population of overweight and obese adolescents. St Laurent et al. [6] investigated the impact of a suspension-training movement on functional movement in children (n = 28, average age = 9.3 years old). Participants were divided into two groups (control and intervention group). After the six-week suspension-training movement program was finished, the intervention group showed better results in functional movement outcomes relative to the control group. Authors of this study suggest that intervention using this kind of training modality could be beneficial for improving functional movement outcomes. Wright et al. [7] examined the impact of fundamental movement training on functional movement outcomes in physically active children (n = 22, average age = 13.4). Participants were divided into two groups, where the intervention group was included in the training that focused on movement quality (weekly 4 × 30-min session) and participants from the control group were involved in multisport activity. Interestingly, results showed that short-term intervention focusing on movement quality did not have an effect on functional movement parameters in physically active children compared to the control group. #### 4.3. Physical Activity, Weight Status, and Functional Movement among Adolescent Populations Although PA level and FM have critically important roles for general health of children, to date, only three studies appear to have examined relations between FM, weight status, and PA level among adolescents [8–11]. Study performed in Moldova investigated the relationship between FM, core strength, posture, and body mass index (BMI) [8]. Researchers collected data from 77 children, from 8 to 11 years old with an average BMI value of 16.4. Mitchell et al. reported the average total FMS score of 14.9 [8], which is slightly higher than the study done by Abraham et al. [2]. The results of this study showed that static posture and BMI are not related to FM. Additionally, researchers did not find a correlation between posture and FMS total score. These results are obvious since posture in this study was assessed in the static position while FMS tests assess movement and dynamic postural stabilization. On the other hand, results showed that core strength was positively related to the total FMS score. The authors of the study concluded that the individual test scores indicate that none of the test items were too difficult for the children, which means that the same tests can be directly used in clinical practice and school classes with school-aged children. An interesting study done by Duncan et al. [9] examined the association between FM and overweight and obesity in British children. Data were obtained from 90 children, 7–10 years old. After BMI was determined, children were classified as normal weight, overweight, or obese according to international official guidelines. The results for total FMS score for normal weight children was 14.7, for overweight 12.2, and for obese children 9.0. Duncan et al., showed that total FMS score was negatively correlated with BMI. Additionally, the scores in all individual FMS tests were higher for normal weight children compared to obese children. In addition, normal weight children performed better than overweight children in the two tests: deep squat and shoulder mobility. On the other hand, overweight children scored better than obese children in four movement patterns (hurdle step, inline lunge, shoulder mobility, and ASLR). This result puts overweight and obese children in the group of children with increased risk for injury incidence. These are clinically important findings, because over time, dysfunctional movement patterns along with the effect of excess weight and consequently higher load on the joints, can possibly lead to degenerative changes in later life. This research highlights that overweight and obesity are significantly associated with poorer functional movement in children. Findings of this study seems to be contradictory with the results of the study mentioned before [8]. However, in a study done by Mitchell et al., there are few limitations that need to be considered. The authors did not separate participants into three categories (normal weight, overweight, and obese). In addition, 9% of the children were categorized as overweight (with no information about number and percentage of obese children), whereas in study done by Duncan et al. one third of children were classified as overweight/obese [9]. This limitation can potentially lead to opposite results and limited conclusion, and therefore can minimize the importance of potential relations between higher values of the BMI and suboptimal functional movement patterns in children. In this research field, one more interesting study was performed by the same authors [10]. Duncan and Stanley investigated relations between weight status, physical activity level, and functional movement in British children. This study was performed on 58, 10–11 year old children. The results showed that the total FMS score was negatively correlated with BMI and positively related to PA level. Normal weight children scored significantly better for total FMS score compared to children classified as overweight/obese. The mean of total FMS scores was 15.5 for normal weight children and 10.6 in overweight/obese children. Duncan and Stanley explained these results through few possible mechanisms. These authors suggested that deficits in FM could exist prior to being overweight. They pointed out that: "Excess weight and functional prowess are the results of natural selection since children who are functionally limited will remain inactive and will not develop optimal functional movement patterns that underpin performance to the same level of mastery as children without functional limitation." [10]. Additionally, the authors discussed that children who are not functionally limited may more likely enjoy PA, and thus, engage in more regular practice of functional movement patterns that underpin performance. Looking altogether, the results presented in these studies support the need for interventions to increase level of physical activity and improve functional movement in overweight and obese pediatric populations. Garcia-Pinillos et al. [11] examined relations between functional movement patterns and weight status in children aged between 6 and 13 years old (n = 333). Results of this study show that weight status is moderately negatively correlated with total FMS score. In addition, overweight and obese children showed poorer functional movement compared to normal weight children. These results are consistent with the study done by Duncan et al. In addition, significant differences were found between normal weight, overweight, and obese children in lower-extremity movement patterns (deep squat, hurdle step, in-line lunge) and flexibility tests (shoulder mobility, straight leg-raise), but also in trunk stability pattern (push-up). This research revealed that girls outperformed boys in tests that require flexibility and balance, while boys outperformed girls in stability tests which support previous findings in context of sex dimorphism in individual functional movement patterns [2]. The information presented in the paragraphs above are essentially important for the practice of physical education teachers, coaches, and other professionals who work with pediatric populations. Optimal level of PA and optimal FM in children can reduce the risks of orthopedic abnormality arising from suboptimal movement patterns in later life. Since suboptimal movement patterns and low PA level could predispose children to a higher risk of the injury incidence, practitioners should consider functional movement interventions. It is the author's opinion that this population needs specific exercises that address suboptimal movement patterns first, and then exercises targeting weight status to minimize risk of high-load exercise on the skeletal system in the pediatric population. #### 4.4. Maturation and Functional Movement among Athletic Populations of Adolescents To date, a number of studies investigated maturation effects in the average and athletic population of adolescents. However, only few studies appear to have examined the relationship between maturity and functional movement patterns [22–25]. These studies included only the athletic adolescent population. Within this paragraph, the author will briefly provide a review of current evidence and conclusions about maturation effects on functional movement among athletic adolescents. A study done by Portas et al. investigated the effect of maturity on functional movement screen scores in elite, adolescent soccer players [22]. The authors showed that maturity has substantial effects on FMS performance. Although this research highlights that findings are relevant only to those analyzing movement of soccer players, the authors of the mentioned study concluded that FMS assessment appears to be invalid for practical usage for very young players. Paszkewicz et al. compared functional and static evaluation tools among adolescent athletes [23]. The authors of this study compared FMS scores and Beighton and Horan joint mobility index (BHJMI) scores among pubescence in adolescent athletes. Based on the results of the modified pubertal maturation observational scale, the authors separated subjects into three groups: prepubescent, early-pubescent, and postpubescent groups. The researchers revealed a main effect for FMS scores across pubertal groups, but not in BHJMI composite scores. The postpubescent participants had higher FMS scores compared with the prepubescent participants and the early-pubescent athletes. Additionally, the results of this study did not confirm any correlation between FMS composite scores and BHJMI composite scores. The results of this study suggest that the FMS can discriminate between levels of pubescence and detect alterations during the pubertal growth cycle, whereas the BHJMI may not. Lloyd et al. examined relationships between functional movement screen scores, maturation, and physical performance in young soccer players [24]. This study demonstrated that variation of physical performance of youth soccer players could be explained by a combination of both functional movement screen scores and maturation. Wright and Chesterton [25] aimed to investigate differences between individual functional movement patterns at different stages of maturation in young athletes (mean age = 14.1 years, age between 8 and 18 years old) from various sports (field athletics, endurance sport, team sports, combat, and water sports). Participants were categorized in the three distinct maturation groups, participants who were before, at, and after their adolescent growth spurt (peak height velocity (PHV)). The authors found that differences among these groups were greatest in movement patterns that have high demands on stability, which suggests that adolescents potentially develop stability in this period of growth. These findings are consistent with studies mentioned before in this section [22,23]. In addition, results of the push-up test were higher in children who were at growth spurt or after growth spurt, when compared with children before growth spurt. In addition, authors of this study concluded that maturation has no effect on total FMS score. However, it appears that there is no study that investigated the relationship between maturation effects and functional movement patterns among the average adolescent population. Due to the importance of this research field, the author is of the opinion that more studies are necessary in this research field. #### 5. Conclusions This paper gave a detailed description and summarization of the current literature in the field of pediatric PA level, obesity, and maturation related to functional movement. Although there are only few studies in this field of research, the author highlights the importance and health benefits of optimal FM in children, as well as the consequences of dysfunctional movement patterns on the health of the locomotor system. Summary of current evidence suggests that decreased physical activity level is negatively correlated to functional movement in adolescence. Additionally, most studies suggest that weight status is negatively correlated to functional movement patterns, although there is conflicting evidence in this area. Evidence consistently showed that overweight and obese adolescent exhibit poorer functional movement compared to normal weight adolescents. Most of the studies that examined effects of exercise intervention on functional movement improved functional movement outcomes, while one study showed the opposite. It is clear that more research is needed on this topic to establish true intervention effects. In addition, it appears that maturation has effects on functional movement in the athletic population of adolescents. It is therefore important that practitioners consider interventions which develop optimal functional movement alongside physical activity and weight management strategies in children, in order to reduce the risks of injuries and pathological abnormality arising from suboptimal movement patterns in later life. Funding: This work was funded by the Croatian Science Foundation, foundation under the number IP-06-2016-9926 and DOK-2018-01-2328. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).	doab	2025-04-07T03:56:58.855430	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 9 }
0051d55d-461b-414c-a51b-301e45ff4d0c.10	Review The Effects of Alcohol Consumption on Recovery Following Resistance Exercise: A Systematic Review #### Nemanja Laki´cevi´c Sport and Exercise Research Unit, Department of Psychological, Pedagogical and Educational Sciences, University of Palermo, 90144 Palermo, Italy; [email protected]; Tel.: +39-3515881179 Received: 14 May 2019; Accepted: 21 June 2019; Published: 26 June 2019 Abstract: Background: The aim of this manuscript was to describe the effects of alcohol ingestion on recovery following resistance exercise. Methods: A literature search was performed using the following database: Web of Science, NLM Pubmed, and Scopus. Studies regarding alcohol consumption after resistance exercise evaluating recovery were considered for investigation. The main outcomes took into account biological, physical and cognitive measures. Multiple trained researchers independently screened eligible studies according to the eligibility criteria, extracted data and assessed risk of bias. Results: A total of 12 studies were considered eligible and included in the quantitative synthesis: 10 included at least one measure of biological function, 10 included at least one measure of physical function and one included measures of cognitive function. Conclusions: Alcohol consumption following resistance exercise doesn't seem to be a modulating factor for creatine kinase, heart rate, lactate, blood glucose, estradiol, sexual hormone binding globulin, leukocytes and cytokines, C-reactive protein and calcium. Force, power, muscular endurance, soreness and rate of perceived exertion are also unmodified following alcohol consumption during recovery. Cortisol levels seemed to be increased while testosterone, plasma amino acids, and rates of muscle protein synthesis decreased. Keywords: strength; training; muscle mass; muscle function; performance #### 1. Introduction Resistance exercise (RE) is a commonly practiced modality of physical exercise used by both amateurs and elite athletes [1]. RE is a type of exercise that has gained a lot of interest over the past two decades, specifically for its role in improving athletic performance by developing muscular strength, power and speed, hypertrophy, local muscular endurance, motor performance, balance, and coordination [2]. While non-athletes use it to simply develop muscular physique, professional athletes engage in RE to enhance their athletic capabilities in various sports [3]. Variables such as exercise intensity, exercise frequency, load, number of sets and repetitions, rest periods and training volume can be manipulated in order to maximize RE induced effects in terms of muscle hypertrophy and strength [4]. Physiological and psychological constraints leading to a reduction in physical or mental performance can be classified as fatigue which is a phenomenon that has protective role in human physiology [5]. Exercise is a potent stimulus with respect to altering homeostatic variables which triggers adaptive reactions that counter the metabolic changes and repair the structural damage caused by the previous training session [5]. The stressful effects of RE can temporarily impair athlete's performance [6]. Therefore, the speed and quality of recovery are absolutely essential for the high performance athlete and, if done correctly, optimal recovery can lead to numerous benefits training and upcoming competition [7]. The main purpose of recovery is to restore physiological and psychological processes, so that the person engaging in vigorous exercise can repeat training sessions at an appropriate level [7]. It is also typically dependent on the nature of the exercise performed and any other outside stressors that the athlete may be exposed to [7]. Certainly, one of the unnecessary stressors during recovery phase is alcohol (ALC) consumption [8, 9]. Worldwide, alcohol is the most commonly used psychoactive drug; it is estimated that each adult person consumes, on average, about 4.3 L of pure alcohol per year [10]. In the current era, consumption of alcohol is increasing exponentially in Western society [11–13] and it is common knowledge that alcohol can permeate virtually every organ and tissue in the body, resulting in tissue injury and organ dysfunction [14]. Alcohol consumption results in hormonal disturbances that can disrupt the physiological ability to maintain homeostasis and eventually can lead to various disorders, such as cardiovascular diseases, reproductive deficits, immune dysfunction, certain cancers, bone disease, and psychological and behavioral disorders [14]. In terms of post exercise recovery, acute alcohol ingestion reduces muscle protein synthesis in a dose-and time-dependent manner, after the cessation of exercise stimulus [8]. Alcohol does this mainly by suppressing the phosphorylation and activation of the mTOR pathways, the crucial kinase cascade regulating translation initiation [8,15]. Concomitantly, alcohol increases the expression of muscle specific enzymes that are up regulated by conditions that promote skeletal muscle atrophy [8,16]. Emerging research provides new insights into the effect of alcohol consumption on post-exercise muscle recovery but more research is needed to determine how this relationship exists and establish the physiological mechanisms governing this response. Therefore, the aim of this review is to understand the effects of alcohol consumption during recovery, on muscle function, following RE. #### 2. Materials and Methods Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement has been used to structure this manuscript. #### 2.1. Inclusion and Exclusion Criteria Studies that meet the following criteria will be included or excluded in this systematic review. #### 2.2. Eligibility Criteria When it comes to eligibility criteria, only articles written in English language and published in peer-reviewed journals have been considered during the search. There was no limit on publication date when it comes to article eligibility. Different formats of publications such as reviews, meta-analysis, abstracts, citations, scientific conference abstracts, opinion pieces, books, book reviews, statements, letters, editorials, non-peer reviewed journal articles and commentaries have been excluded. With respect to intervention, publications were included only if they used a specific measure of performance or biomarker, that considered recovery following RE and alcohol intoxication. Articles exploring recovery after endurance type of training have been eliminated. #### 2.3. Participants All the analyzed participants were adults to whom an ALC intervention was administered following a bout of RE. Children were not considered for analysis. There was no limitation when it comes to age, gender, number of participants, and duration of intervention or follow up period. Furthermore, there was no limitation when it comes to training status. #### 2.4. Interventions The interventions described in the eligibility criteria will be included in this review. The interventions aimed to understand the effects of ALC on biological, physical and cognitive measures following RE. According to the nature of the review different methodological approaches which evaluate similar outcomes will be considered. #### 2.5. Comparators Comparators will be control groups (people not consuming ALC [NO-ALC]) if present or if cross-over designs will be adopted the intervention groups will act as controls after each wash-out period. #### 2.6. Outcomes The primary outcome will be to understand the effects of ALC compared to the NO-ALC intervention. Such findings will be applied to all the biological, physical and cognitive measures retrieved. #### 2.7. Search Strategy We used EndNote v. 8.1 software (Clarivate Analytics, Jersey, UK) for the article search. The papers have been collected through PubMed (NLM), Web of Science (TS), and Scopus using the string: (("alcohol" AND "exercise\" and "recovery\"; "ethanol" AND "exercise" AND "recovery"; "alcohol\" AND "resistance training" AND "recovery"; "ethanol" AND "resistance training\" AND "recovery"; "alcohol\" AND "strength\" AND "recovery"; "ethanol" AND "strength" AND "recovery"; "alcohol\" AND "training" AND "recovery"; "ethanol" and "training" and "recovery")). #### 2.8. Selection of Study Objects* The article screening was carried out in a three-step process: title reading, abstract reading and full text reading, respectively. If any disagreements were noticed between the two investigators, a third one considered the current process independently and discussed the decision with the other investigators. Furthermore, investigators were not blinded to the manuscripts, study title, authors, or associated institutions during the selection process. Both qualitative and quantitative articles were included in the review. The screening processes have been summarized via PRISMA flow diagram (Figure 1). #### 2.9. Risk of Bias Risk of bias for the included studies was assessed through Downs and Black checklist [17]. This tool is useful when evaluating the quality of original research articles in order to synthesize evidence for public health purposes. This checklist contains 27 'yes'-or-'no' questions over five different domains. It offers both an overall score for study quality and a numeric score out of a possible 32 points. The five domains contain questions about study quality, external validity, study bias, confounding and selection bias, and power of the study. Two independent researchers have completed the Downs and Black checklist of all included articles to determine the quality of each study. The maximum score a study can receive is 32, with higher scores indicating better quality. The studies were then divided into groups and marked as 'high quality' (score 23–32), 'moderate quality' (score 19–22), 'lower quality' (score 16–18) or 'poor quality' (<14) (Supplementary File S1 and S2). Kendall Tau correlation coefficient statistical method was used to determine inter-rater reliability. We used R statistical software (Bell Laboratories, Murray Hill, NJ, USA) version 3.6 to perform this analysis. Quality of evidence was determined by the study design and by Downs and Black score. Figure 1. Prisma Flow Diagram #### 2.10. Data Synthesis The critical information acquired from the included articles was extracted into Microsoft Excel for Macintosh, version 14.0 (Microsoft Corp, Redmond, WA, USA) spreadsheet. The most important characteristics of the studies, as author name and publication year, sample size, aim, alcohol dose, how this was mixed and administered, the study measures, the RE protocol adopted, the study design and the outcomes have been delineated in the tables while certain specifics about the particular study were described in a narrative manner. #### 3. Results From a preliminary title and abstract search, a total number of 471 studies have been identified in the three screened databases. After the application of inclusion criteria on each article's title and abstract, 63 records were considered eligible. Duplicates were removed leaving 24 studies for full text screening. After full text screening, two additional studies from the relevant bibliography have been added. Of the 24 studies analyzed, 10 were included for the final synthesis. Therefore a total number of 12 studies were included in the qualitative synthesis of this review article. The process of article inclusion has been synthesized in Figure 1. Risk of bias assessment was finalized through Downs and Black checklist for all included studies (Supplementary File S1 and S2). The mean score was 19 (range = 12–28). After performing the inter-rater reliability test via Kendall Tau analysis we detected the score of 0.46 which can be classified as moderate but significant (p-value 0.026). As previously stated, after comprehensive screening, files were split into different quality categories in accordance to predetermined quality criteria (Supplementary File S1 and S2). In order to evaluate the effects of alcohol consumption on recovery following RE, the results have been summarized into three categories: (1) biological and (2) physical measures and (3) cognitive function. Of the retrieved studies, 10 took into account at least one biological measure [9,18–26], 11 took into account at least one physical measure [9,18–25,27,28] and cognitive function only one [24]. The retrieved biological measures include creatine kinase (CK) [18–25], heart rate [19,26], lactate [19,26], blood glucose [9], urine measures [24], cortisol [19,21,24,26], testosterone [19,21,24,26], estradiol [26], sexual hormone binding globulin (SHBG) [21,26], leukocytes and cytokines [19,21,22], C-reactive protein (CRP) [24], plasma amino acids [9], intracellular signaling proteins and rates of muscle protein synthesis (MPS) [9] and calcium (Ca2<sup>+</sup>) [25]. The physical measures include force [18,20–25,27,28], power [19,21,24,28], muscular endurance [25], soreness [18,20,22,23,28] and rate of perceived exertion (RPE) [19,24,26]. The cognitive measures included a modified version of the STROOP test which evaluated time and accuracy of each response for congruent and incongruent stimuli. The alcohol dose provided to the participants in the included studies ranged between 0.6g/kg to 1.5g/kg. As defined by Kalinowski and Humphreys [29] a standard drink equals to 10 g of alcohol. Therefore, the alcohol dose provided to the participants, if we consider a man of 70kg, equals to 42 to 105 g of alcohol (4.2 to 10.5 standard drinks), which corresponds to 3 bottles of beer of 330 mL at 5% alcohol or a 370mL bottle of spirit at 37.5% alcohol, respectively. All the included studies adopted a cross-over research design. A summary of the retrieved studies is shown in Table 1. #### 3.1. Biological Measures #### 3.1.1. Creatine Kinase Eight studies included CK measurement [18–25]. All the retrieved studies showed that CK increases following each RE protocol in both ALC and NO-ALC conditions showing a time interaction with RE. However, when analysing interaction with the different conditions, no differences were shown between the ALC and NO-ALC condition for none of the retrieved studies. Clarkson et al. [20], have also correlated peak CK activity from the ALC and the NO-ALC condition and found a high correlation coefficient (r=0.95), whereas Paulsen et al. [25] have also stratified the findings for man and women finding again no differences between the two groups neither for time or treatment assessment. Such results, as also highlighted by each author of the included studies, demonstrate that ALC cannot be considered as a modulating factor for CK following RE and that the increases of CK are the result of muscle damage following the exercise bouts. #### 3.1.2. Heart Rate Two studies included measures of heart rate [19,26]. In both studies heart rate increased following the exercise intervention, however no difference was shown between the ALC and the NO-ALC condition. #### 3.1.3. Lactate Two studies included measures of lactate [19,26]. In both studies lactate increased following the exercise intervention, however no difference was shown between the ALC and the NO-ALC condition. #### 3.1.4. Blood glucose Only Parr et al. [9] evaluated the concentration of blood glucose. It has to be noted that Parr administered a concentration of alcohol in conjunction with either CHO or PRO. The results highlight a significant time and treatment interaction. Blood glucose concentration increased 0.5 and 4.5 h post intervention in the ALC-CHO group but not in the ALC-PRO or NO-ALC groups. Such findings demonstrate that blood glucose is affected by CHO but not ALC during recovery after RE. differences in leukocytes. any group. Table 1. Descriptive characteristics of the retrieved studies. was observed in the ALC group. globulin; RM= Branched Chain amino acids; FAI= Free androgen index. Repetition maximum; CRP= C-reactive protein; CHO= carbohydrate; PRO= Protein.; AA= Amino Acids; PPO= Peak power output; EAA = Essential amino acids; BCAA= #### 3.1.5. Urine Measures Only Murphy et al. [24] included urine measures. Post-intervention urine output, nude mass and urine-specific gravity were measured. No differences were found for nude mass and urine-specific gravity between conditions. The ALC group had an increased total volume output overnight when compared to the NO-ALC group. #### 3.1.6. Cortisol Four studies included measures of cortisol [19,21,24,26]. In the study of Barnes et al. [19] the cortisol levels increased after 12h after treatment in both conditions, after which at 24h returned to baseline levels. A second rise in cortisol was seen at 36h under the ALC condition but not in the NO-ALC condition. Haugvard et al. [21] showed that no differences were shown between the two conditions at any specific time point after the intervention (12 and 24 h post-treatment). However, if the 12 and 24 h cortisol values were combined and averaged, these resulted to be significantly elevated only in the ALC condition at 24 h after the intervention. Murphy et al. [24] showed that a significant decrease post-match was followed by a significant increase at 16h post intervention. No difference was found in the levels of cortisol between the two interventions. However, a large effect size was found between the %change from 2 to 16 h post-match for the increase in cortisol response after ALC consumption. Vingren et al. [26] found that cortisol levels were not affected by ALC post intervention. Cortisol was elevated immediately after, after 20, 40, 60, 120, 140 and 300 min post-intervention in both ALC and NO-ALC conditions. #### 3.1.7. Testosterone Four studies included measures of cortisol [19,21,24,26]. In the study of Barnes et al. [19] no difference in the testosterone levels compared to baseline ware seen at any time point after the intervention in either two conditions (12-24-36 and 48 h after the intervention). In the study of Haugvard et al. [21] the levels of testosterone were not altered across trials neither for the ALC and the NO-ALC condition. Calculated free testosterone (testosterone/SHBG multiplied by a factor of 10) was not different between trials. However, if the measures at 12 and 24 h after the intervention were combined and averaged the levels of testosterone resulted to be lower only in the ALC condition after 24 h. Murphy et al. [24] showed that a reduction in testosterone was present after 2 h post-match followed by a significant increase 16h post-match. However, no differences were shown between the testosterone levels for the two conditions neither between %changes from 2 to 16 h post-match. Vingren et al. [26] found a significant effect for treatment for testosterone in which the levels were increased immediately and 140 and 300 min after the intervention for the ALC group, whereas it appeared to be decreased in the NO-ALC group between 60 and 300 min post-intervention. Free testosterone also seemed to be increased between 60 and 300 min post-intervention for both conditions. #### 3.1.8. Estradiol Only one study included measures of estradiol after ALC consumption [26]. The study indicates that the levels of estradiol were elevated immediately after and between 20 and 40 min after the intervention, when compared to baseline measures, in both groups, with no significant differences between the ALC and the NO-ALC group. The results underlie that ALC has no effect on estradiol during recovery from RT. #### 3.1.9. Sexual Hormone Binding Globulin Two studies included measures of SHBG [21,26]. In none of the included records the levels of SHBG seemed to be altered by ALC intake, neither by the acute bouts of exercise proposed by the two authors. ALC does not seem to be a modulating factor for SHBG following RE. #### 3.1.10. Leukocytes and Cytokines Three of the included studies included measures of leukocytes and cytokines [19,21,22]. Barnes performed analysis of total and differential leukocytes and found that total, neutrophil and monocyte concentration increased after the intervention but decreased to baseline values after 12h. However, no difference was present between the two conditions. Haugvard et al. [21] found no difference between conditions regarding the white blood cell, neutrophils or monocytes count. Following RE both conditions showed a sub-clinical leucocytosis 1h post-exercise. Levitt et al. [22] analysed inflammatory markers in women post-exercise, in particular TNF-α, IL-1β, IL-6, IL-8 and IL-10 before, at 5, 24 and 48h post-intervention.IL-10, IL-8 and TNF-α increased after the intervention in both groups. IL-6 and IL-1β remained unchanged over time for both conditions. No differences for cytokine was present between the ALC and the NO-ALC condition. ALC doesn't seem to affect neither leukocytes nor cytokines after RE during recovery. #### 3.1.11. C-reactive Protein C-reactive protein was evaluated only in the study of Murphy et al. [24], in which however no significant difference was highlighted neither regarding time, when data was compared to baseline, neither regarding condition, when ALC and NO-ALC where compared. The findings indicate that post-match alcohol consumption did not unduly affect CRP markers of damage. #### 3.1.12. Plasma Amino Acids Plasma amino acids (AA) have been included only in the study of Parr et al. [9], who evaluated EEA, BCAA and leucine at 0, 1, 2, 4, 6 and 8 h after alcohol consumption following RE. It has to be noted that Parr administered a concentration of alcohol in conjunction with either CHO or PRO. The results were then compared to a control group who did not ingest ALC but consumed a single dose of 25 g of whey protein. A significant effect for time and treatment were found. At all-time points the NO-ALC group had significantly higher levels of essential AA (EEA), branched chain AA (BCAA) and leucine compared to the ALC-PRO group. Both groups (the NO-ALC and the ALC-PRO) had at all-time points significantly higher levels of AA compared to the ALC-CHO group. No difference in the levels of AA compared to baseline was shown, at any time point, in the ALC-CHO group. Leucine, EEA and BCAA were elevated compared to baseline at 1 and 6 h post-ALC ingestion for the NO-ALC and ALC-PRO group. The data from the study of Parr et al. [9] indicates that ALC alone does not influence the levels of plasma AA, however can be a factor that limits the rise of blood concentration of AA following protein consumption. #### 3.1.13. Intracellular Signaling Proteins and Rates of Muscle Protein Synthesis mTOR, p70S6K, eEF2, 4E-BP1, AMPK, MuRF-1 mRNA and fractional synthetic rate of myofibrillar protein synthesis were analysed in the study of Parr et al. [9]. ALC and NO-ALC consumption modalities have been described in the previous subsection. mTORSer2448 phosphorylation was higher in all groups at 2h post treatment. However, mTOR phosphorylation in the NO-ALC group was higher than the ALC-CHO (76%) and ALC-PRO (54%) group at 2 and 8 h post-exercise. p70S6K phosphorylation was greater after 2h post-exercise compared to baseline only in the NO-ALC and the ALC-PRO groups. No differences were shown for the ALC-CHO group. eEF2 phosphorylation decreased below rest values at 2 and 8 h in the ALC-CHO and ALC-PRO groups. No differences were shown for the NO-ALC group at any time point. No differences for time and condition were shown for 4E-BP1Thr37/<sup>46</sup> or AMPKThr172 phosphorylation. There were increases above rest in MuRF-1 mRNA at 2 h post- intervention with no differences between treatments. All values returned to baseline at 8h post- intervention. Fractional synthetic rate of myofibrillar protein synthesis were increased above baseline for all groups from 2 to 8 h post-intervention. However, a hierarchical reduction was shown when data was compared to the NO-ALC group in the ALC-PRO (-24% compared to NO-ALC) and ALC-CHO (−38% compared NO-ALC and −18% compared to ALC-PRO) groups. Data suggests that ALC consumption impairs the response of muscle protein synthesis during recovery despite optimal nutrient provision. #### 3.1.14. Calcium Only one study has evaluated the effects of ALC on Ca2<sup>+</sup> via blood sampling [25]. The authors report that the Ca2<sup>+</sup> levels were similar before exercise for both conditions. A decrease of approximately 2% was observed following the exercise bouts. A further decrease was observed in the ALC condition, and the difference with the NO-ALC condition was significant only after the strength evaluation. Hypocalcaemia was not induced by ALC and no differences were shown for resting free Ca2<sup>+</sup> levels indicating that free Ca2<sup>+</sup> concentrations were not affected by alcohol per se. #### 3.2. Physical Measures #### 3.2.1. Force Nine studies have examined the effects of post-exercise ALC consumption on force [18,20–25,27,28]. McLeay et al. [23] evaluated maximal isometric, concentric and eccentric muscular contractions of the quadriceps femoris using an isokinetic dynamometer for both lower limbs using one lower limb as control. A significant difference between lower limbs was present post-treatment (exercised vs. non-exercised lower limb) up to 60 h post-exercise regarding maximal isometric tension, concentric and eccentric torque but no difference was observed between the ALC and the NO-ALC condition. Barnes et al. [18,27] in both studies evaluated isometric, concentric and eccentric contractions of the quadriceps muscles of both lower limbs using one lower limb as control. Isometric tension was measured at 75◦ of knee angle. Concentric and eccentric torque was measured at an angular speed of 30◦/s. In both studies a decrease in performance was seen in both the ALC and NO-ALC groups over time in the exercised lower limb for all the evaluated measures (isometric and eccentric peak torques as well as for isometric, concentric and eccentric average peak torques). A greater decrease in performance was however seen in the ALC group in the first study [27] at 36h post-intervention (isometric and eccentric peak torques were reduced 39 and 44% compared to pre-exercise measures, respectively, with ALC whereas losses of 29 and 27% for the same measures in the NO-ALC group. Average peak torque was reduced by 41% (isometric), 43% (concentric) and 45% (eccentric) with ALC compared to 29, 32 and 26% with NO-ALC groups, respectively), while no differences were seen between 36 and 60h post-intervention. In the second study [18], except for average peak isometric torque, all measures were different between interventions with the greatest decrements observed in the ALC group. Greatest decreases in peak torque were observed at 36 h with losses of 12%, 28% and 19% occurring in the NO-ALC group for isometric, concentric and eccentric contractions, respectively. Peak torque loss was significantly larger in ALC with the same performance measures decreasing by 34%, 40% and 34%). Levitt et al. [22] measured peak torque for the knee extension exercise on each lower limb using an isokinetic dynamometer. The same assessment procedure used by Barnes et al. [18,27] was adopted. A reduction post-intervention was found for peak isometric, concentric and eccentric torque between the exercised and not-exercised lower limb, but no difference was found between the ALC and NO-ALC condition immediately post nor at 24 and 48 h post-intervention. Poulsen et al. [25] evaluated isokinetic muscle strength of the dominant knee extensors and non-dominant wrist flexors. No differences in isometric strength was observed immediately post, 4, 24 or 48 h post intervention, neither regarding time, when compared to baseline, neither regarding ALC condition. Murphy et al. [24] measured peak MVC of the knee extensors and found that a significant reduction compared to baseline was evident at all measured time points (2 and 16 h post-intervention), but no differences were present between the ALC and NO-ALC group. Haugvad et al. [21] have also measured isometric MVC of the knee extensors and the results reported by the authors showed stable values in all analysed conditions (Low ALC dose, High ALC dose and NO-ALC). A decrease immediately after performance and a recovery from immediately after to 12 and 24 h post intervention was seen in all groups, with no significant differences between trials. Clarkson et al. [20] measured isometric strength of the elbow flexors and the results are similar to those of Haugvad et al. with a reduction immediately post-exercise but no difference between conditions. The level of isometric strength returned to baseline 5 days postintervention. Except for the studies of Barnes et al. no differences seem to be present following ALC consumption on force during recovery following RE. It has to be noted that the ALC dose provided by Barnes et al. is of 1g/kg, which is neither the minimum or maximum dose provided across the studies. #### 3.2.2. Power Four studies [19,24,28,30] included measures of power following ALC consumption and RE. Barnes et al. [19] have included measures of counter movement jump (CMJ) and horizontal power output (HPO). HPO did not vary neither over time neither regarding condition. CMJ instead showed a significant time x treatment effect, where a decrease in jump performance was observed after 24 and 48 h post-intervention only in the ALC group. However, the authors underline that the decrements seen in performance are trivial as the decrease in the jumping performance of the CMJ was a mean value of 12 cm. Murphy et al. [24] have included a measure of CMJ over time, where each participant was required to perform 10-maximal repeated CMJs. The results of Murphy et al. do not show any difference neither regarding time neither regarding condition. Levitt et al. [28] have included measures of vertical power, and similarly to Barnes et al., the reported measures of power show a time effect, with a reduction in vertical power output after 24 and 48 h post-intervention, but no effect regarding condition. Haugvad et al. [21] included a measure of squat jump performed without any counter movement on a force platform. Jump height was calculated for analysis. Jump height was reduced immediately after and 12 h post-intervention in all groups (low-ALC, high-ALC and NO-ALC condition). However, no difference between any group was present. ALC doesn't seem to have an effect on power output, at least in the 48 h following its consumption. #### 3.2.3. Muscular Endurance Maximal isokinetic muscular endurance was calculated for the dominant knee extensors and non-dominant wrist in the study of Poulsen et al. [25] using an isokinetic dynamometer. Thirty maximal reciprocal movements were performed at a velocity of 180◦/s without any rest interval. Subjects were instructed to exert maximal effort in every single movement and not to economise the muscle exertion. An endurance index was calculated defined as the mean-peak torque of the last five repetitions as a percentage of the mean-peak torque of the first five repetitions. The results obtained by the authors show no differences in muscular endurance 4, 24 and 48h after treatment neither after ALC intoxication neither in the NO-ALC group. No changes were evident neither in the leg extensors neither in the wrist flexors or between the endurance index for both conditions for both muscle groups. The results were also stratified according to gender and similar findings were achieved. #### 3.2.4. Soreness Five studies included measures of soreness [18,20,22,23,28]. Barnes et al. [18] evaluated soreness by asking each participant at different time points their levels of soreness by giving a value from 0 (no pain) to 10 (worst possible pain). Soreness was rated while stepping up (concentric muscular contraction) onto a 40 cm box and lowering into a squatting position. Clarkson et al. [20] evaluated soreness by questionnaire, measured for the forearm flexor muscles, using a scale of 1 to 10. Levitt et al. [22] measured soreness applying on the vastuslateralis, in three different points along the muscle belly, a pressure of 35N. Each participant rated the pain giving a value from 0 to 10. In a subsequent study Levitt et al. [28] evaluated pain by asking the participants to self-report their level of pain using a scale from 0 to 5. McLeay et al. [23] used the same protocol as above described in the study of Barnes et al. [18]. All the retrieved records show a time effect for muscle soreness related to the intervention protocol, with increases over a period of 24 and 48 h after the training intervention, with no differences between the ALC and the NO-ALC condition. The results indicate that RT is a factor responsible to increase muscle soreness between 24 and 48 h post training, whereas ALC consumption is not. #### 3.2.5. Rate of Perceived Exertion Rates of perceived exertion were measured in three of the retrieved studies [19,24,26]. The study of Vingren [26], was the only one evaluating RPE before and after a single session of static RE. Barnes and Murphy [19,24] evaluated RPE before and after a rugby match. In particular Murphy et al. evaluated RPE after a competitive rugby league game, whereas Barnes et al. after a simulated rugby match. The results of Vingren and Murphy highlight a significant time effect, with increases of RPE after the RT and the rugby league game, but no significant differences between the RPE of the ALC or the NO-ALC groups. The results of Barnes et al. are in line to those of the previous authors regarding the time effect of RPE following the exercising protocol, however a difference was shown between conditions. The ALC group reported lower levels of RPE at the end of the third quarter of the simulated game, compared to the NO-ALC group at the same time measurement. Despite the significant results, the mean difference between the two conditions is very small (ALC=15.2 ± 1.6; NO-ALC=16.5 ± 1.2) and not present at any other time point. #### 3.3. Cognitive Function Cognitive function was assessed only in the study of Murphy et al. [24] through a modified version of the Stroop test. This test of cognitive function was a computer-based program requiring subjects to react to repeated color and word stimuli. The program analyzed response time and accuracy for congruent and incongruent stimuli. Measures of cognitive function were recorded before, immediately post, 2 and 16 h intervention. The results provided by the authors show no difference over time for cognition test time, congruent reaction time, or incongruent reaction time. However, the time required to complete the cognition test significantly increased in the ALC compared to the NO-ALC group and a large ES was shown for increased cognition test time, congruent and incongruent reaction time in the ALC group compared to the NO-ALC group. The findings indicate that ALC consumption impairs cognitive function during recovery, which may be a negative factor in sports where decision making processes, speed and quality of responses to visual stimuli (especially team sports) are essential. #### 4. Discussion By evaluating the effects of alcohol consumption on recovery following RE from biological, physical and cognitive perspective, we have been able to provide a comprehensive description of the multifactorial nature of alcohol consumption. Indeed, alcohol consumption is a common occurrence in the general population on global scale and it is a phenomenon that has not been explored in depth when it comes to post-exercise recovery, even more so in RE post-exercise recovery. The main findings highlight that ALC cannot be considered as a modulating factor for the majority of the retrieved biological measures. In fact, creatine kinase, heart rate, lactate, blood glucose, estradiol, sexual hormone binding globulin, leukocytes and cytokines, C-reactive protein and calcium do not seem to be modified following ALC consumption during the acute recovery phase post-resistance exercise. Only cortisol levels seem to be increased, conversely testosterone, plasma amino acids, and rates of muscle protein synthesis decreased. When considering the retrieved physical measures force, power, muscular endurance, soreness and rate of perceived exertion also seem to be unmodified following alcohol consumption during recovery. The general findings therefore highlight that muscle function is not altered by alcohol consumption following exercise bouts, however the altered endocrinological asset regarding cortisol and testosterone and the consequent suppressed rates of muscular protein synthesis and reduced circulating levels of amino acids, suggest that long-term muscular adaptations could be impaired. A trend of heart rate increase following the exercise intervention has been detected, however no difference was shown between the ALC and the NO-ALC condition. This conclusion raises different concerns since alcohol acts as a diuretic and it contributes to faster elimination of water content from the bloodstream, leading to increased viscous blood plasma which is harder to pump and deliver to the body tissues [31]. The heart has to adapt to these conditions to increase the cardiac output. There seems to be a dose response relationship between the alcohol consumption and heart rate i.e., there is a positive correlation between alcohol consumption and heart rate response [32]. Consequently, this has the potential to alter individual RPE [33]. This latter parameter also seems to not be influenced by alcohol consumption. Only one of the retrieved studies has shown there was a difference between the ALC and NO-ALC group with the ALC group showing less perceived exertion compared to the NO-ALC group. As previously stated alcohol acts as a diuretic and thus can explain why in the study of Murphy at al [24] the ALC group had an increased total volume output overnight when compared to the NO-ALC group. One of the most interesting findings of this review was found in the study by Parr et al. [9] who demonstrated that blood glucose is affected by CHO but not ALC during recovery after RE. This is in alignment with findings of Lustig [34] who claims that toxic effects of alcohol are very similar to excessive sugar exposure mainly for its fructose content. Even though fructose does not show the same acute toxic effects of ethanol, it encompasses all the chronic hazardous effects on long-term health [34]. Creatine kinase was also unmodified by ALC consumption. Such enzyme which is present in the muscles, when detectable in the peripheral circulation, is commonly used as a measure of muscle damage [8]. None of the authors which reported measures of CK showed differences between groups, instead correlations were established between the ALC and the NO-ALC condition. ALC cannot be considered as a modulating factor for CK following RE and the increases of CK shown are the result of muscle damage following the exercise bouts. Neither leukocytes nor cytokines seem to be changed following alcohol consumption, which means that the inflammatory response is not modulated by alcohol consumption. Such is a controversial finding because as reported by different authors [35,36] alcohol abuse not only increases inflammation but also alters the immune function of the body. Probably healthy individuals, who regularly exercise, as those included in each study of this review, do not express altered inflammatory or immune function following a single acute alcohol intoxication. Same trend is shown by CRP, which confirms that muscle damage and inflammation are not dependent, in the analyzed population, from the ingestion of alcohol [24]. Such findings may also explain why perceived soreness was not different between the ALC and NO-ALC groups analyzed. Cortisol and testosterone levels during post RE when compared between ALC and NO-ALC groups appear to be altered. On average, the participants who consumed ALC expressed higher levels of cortisol and lower levels of testosterone in comparison to the NO-ALC group. Decreased levels of testosterone and increased levels of cortisol are suggested to be indicative for a disturbance in the anabolic-catabolic balance, which likely leads to decreased recovery and therefore, decreased levels of performance [24,37]. When present in excessive levels, cortisol is an overall catabolic hormone, which decreases lean body and muscle mass and increases energy expenditure [38]. Conversely, testosterone is an anabolic hormone, which may also explain why in the study of Parr et al. mTOR phosphorylation in the NO-ALC group was higher than the ALC-CHO (76%) and ALC-PRO (54%) group at 2 and 8 h post-exercise. In addition, also rates of muscle protein synthesis were higher in the NO-ALC group when compared to those who ingested ALC. However, muscle protein synthesis may also appear decreased because of the decreased plasma levels of AA showed following ALC consumption. These findings can have major implications with regards to the recovery and performance of both non athletes and professional athletes. An acute bout of vigorous RE can result in a transient increase in protein turnover and until feeding, protein balance remains negative [39,40]. Protein ingestion post exercise enhances muscle protein synthesis and net protein balance [41] by increasing myofibrillar protein fraction with RE [42], but as seen in the study of Parr et al. alcohol ingestion after RE has the ability to disrupt this process. Beyond physical aspects, decreased protein synthesis leads to impaired long-term memory in humans [43–45], which can be particularly important in professional athletes who have many cognitive demands with respect to both short and long term memory [17]. In regards to measures of force only study of Barnes et al. [18] showed that following ALC consumption the levels of isometric, concentric and eccentric torque decreased, while other studies in this review that measured force production showed no differences between the ALC and NO-ALC groups during recovery following RE. As depicted in the results section, with respect to muscle function and force, only study by Barnes et al. has shown that moderate consumption of alcohol can amplify the loss of force associated with strenuous eccentric exercise [18]. This particular study detected significant decrements in average peak isometric, concentric and eccentric torques at 36 h post-exercise [18]. Clearly more research is needed since the outcomes among the mentioned studies are quite distinct. All measures of force were assessed from 2 h to 48 h post RT or ALC ingestion and the measures all appear decreased because of the exercise performed. The retrieved measures of performance returned to baseline within 2 days following both ALC consumption and the exercise bouts. Same trend is shown for the other two measures of performance retrieved: power and muscular endurance which decreased following the exercise bouts in both groups with no difference between those who consumed ALC and those who did not. Several limitations have been encountered during the realization of this manuscript. Firstly, a very limited body of evidence was present within each screened database, on the topic of alcohol consumption following bouts of RE, therefore it is not possible to consider such review comprehensive and definitive. Few studies have evaluated in depth biological measures of protein synthesis or specific markers of muscular function. Other important limitation is the timely evaluation of each study. Each included measure was evaluated in a timeframe ranging from 2 h to 48h post exercise or alcohol consumption. Therefore, only acute modifications were evaluated and it was not possible to consider hormonal fluctuations beyond 2 days and their relative effects. Lastly, the total sample size of each study was small ranging between 8 and 19 participants. #### 5. Conclusions Alcohol consumption following resistance exercise doesn't seem to affect the majority of the retrieved biological and physical measures. However, levels of cortisol were increased, and levels testosterone and rates of muscle protein synthesis were decreased, which indicates that long term muscular adaptations could be impaired if alcohol consumption during recovery is consistent. Muscle function doesn't seem to be influenced by alcohol consumption during recovery. Studies with larger cohorts evaluating the effects of alcohol consumption during recovery following resistance exercise are needed to further understand the long-term effects of alcohol ingestion. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2411-5142/4/3/41/s1. Author Contributions: Conceptualization, N.L.; Methodology, N.L.; Writing – Original Draft Preparation, N.L.; Writing – Review & Editing, N.L; Funding: This research received no external funding. Acknowledgments: I would like to thank Kaltrina Feka and Valerio Giustino from PhD program in Health Promotion and Cognitive Sciences at University of Palermo for helping me to do the article search and assess risk of bias. Conflicts of Interest: The author declares no conflict of interest. #### References © 2019 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).	doab	2025-04-07T03:56:58.857040	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 10 }
0051d55d-461b-414c-a51b-301e45ff4d0c.11	Article Acute Cardiometabolic Responses to Multi-Modal Integrative Neuromuscular Training in Children *Avery D. Faigenbaum \, Jie Kang, Nicholas A. Ratamess, Anne C. Farrell, Mina Belfert, Sean Duffy, Cara Jenson and Jill Bush Department of Health and Exercise Science, The College of New Jersey, Ewing, NJ 08628, USA; [email protected] (J.K.); [email protected] (N.A.R.); [email protected] (A.C.F.); [email protected] (M.B.); [email protected] (S.D.); [email protected] (C.J.); [email protected] (J.B.) \* Correspondence: [email protected]; Tel.: +1-609-771-2151 Received: 10 May 2019; Accepted: 21 June 2019; Published: 24 June 2019 Abstract:** Integrative neuromuscular training (INT) has emerged as an effective strategy for improving health- and skill-related components of physical fitness, yet few studies have explored the cardiometabolic demands of this type of training in children. The aim of this study was to examine the acute cardiometabolic responses to a multi-modal INT protocol and to compare these responses to a bout of moderate-intensity treadmill (TM) walking in children. Participants (n = 14, age 10.7 ± 1.1 years) were tested for peak oxygen uptake (VO2) and peak heart rate (HR) on a maximal TM test and subsequently participated in two experimental conditions on nonconsecutive days: a 12-min INT protocol of six different exercises performed twice for 30 s with a 30 s rest interval between sets and exercises and a 12-min TM protocol of walking at 50% VO2peak. Throughout the INT protocol mean VO2 and HR increased significantly from 14.9 <sup>±</sup> 3.6 mL·kg−1·min−<sup>1</sup> (28.2% VO2 peak) to 34.0 <sup>±</sup> 6.4 mL·kg−1·min−<sup>1</sup> (64.3% VO2 peak) and from 121.1 <sup>±</sup> 9.0 bpm (61.0% HR peak) to 183.5 ± 7.9 bpm (92.4% HR peak), respectively. While mean VO2 for the entire protocol did not differ between INT and TM, mean VO2 and HR during selected INT exercises and mean HR for the entire INT protocol were significantly higher than TM (all Ps ≤ 0.05). These findings suggest that INT can pose a moderate to vigorous cardiometabolic stimulus in children and selected INT exercises can be equal to or more metabolically challenging than TM walking. Keywords: Heart rate; interval training; metabolism; oxygen consumption; physical activity; resistance training; strength training; youth #### 1. Introduction A growing number of children and adolescents fail to accumulate at least 60 min of moderate to vigorous physical activity (MVPA) daily [1]. The far-reaching consequences of physical inactivity during childhood and adolescence are a constellation of cardiometabolic, musculoskeletal, and psychosocial risk factors and diseases that are challenging to manage, difficult to treat and costly to individuals and society [2]. Current efforts to increase MVPA in youth with targeted interventions have had only a small effect [3]. Notably, the impact of walking interventions on physical activity behaviors and health-related fitness measures in school-age youth have been limited [4–6]. While walking is a natural form of physical activity that is practical and inexpensive, other types of exercise may be needed to enhance cardiometabolic health, target neuromuscular deficiencies, and increase participation in MVPA. The importance of integrating different types of resistance exercise into youth fitness programs has become particularly important in light of secular declines in measures of muscular strength and power in modern day youth [7,8]. The available evidence supports a link between muscular fitness and physical activity, particularly vigorous intensity physical activity, in children and adolescents [9]. Integrative neuromuscular training (INT) is a type of exercise characterized by intermittent bouts of different strength- and skill-building exercises that are designed to improve fundamental movement skills, increase muscular fitness and prepare participants for exercise and sport activities [10]. INT has emerged as an effective strategy for improving health- and skill-related components of physical fitness in school-age youth, [11–13], and limited evidence suggests that this type of training may also offer cardiometabolic benefits [14,15]. Previous studies investigating the effects of INT on children have found significant improvements in sprinting, running, jumping, throwing and lifting performance following 8 to 10 weeks of training [11,12,16]. However, it is also important to examine the acute cardiometabolic responses to INT because the amount of time youth spend in vigorous physical activity is more strongly associated with positive health outcomes than light or moderate-intensity physical activity [17–19]. An analysis of accelerometer data from a large sample of children found vigorous physical activity was strongly associated with metabolic health whereas associations of light to moderate physical activity were weak to moderate [18]. While research evidence supports the safety, efficacy and feasibility of INT for children [11,12,16], the acute cardiometabolic responses to INT are poorly understood. Researchers examined the acute cardiometabolic responses to a single-mode interval training protocol with medicine balls [14] or battling ropes [15] in children and found that this type of exercise can pose a potent cardiometabolic stimulus. For example, mean heart rate (HR) and oxygen uptake (VO2) values during a 10-min bout of medicine ball interval training ranged from 61.1% to 89.6% of HR peak and from 28.2% to 63.5% of VO2 peak [14]. Similarly, others found that the acute responses to a 12-min session of resistance training or intermittent noncontact boxing in early adolescents could be characterized as "vigorous" and therefore contribute to daily moderate to vigorous physical activity (MVPA) recommendations [20]. Due to the increasing interest in high-intensity interval training and the potential for multi-modal INT to modulate disease risk factors and improve health outcomes in youth [10,21,22], there is strong rational to further examine the acute cardiometabolic responses to INT in youth. Of relevance to the current study, strategic efforts to strengthen and improve physical education and physical activity opportunities with novel and time efficient exercise interventions are needed to increase MVPA in school-age youth and foster a healthy generation [23]. To the authors' knowledge, no previous study has examined the acute cardiometabolic responses to multi-modal INT in children and direct comparisons between INT and traditional exercise interventions such as walking have not been reported. While brisk walking can offer health benefits for children, INT has been found to enhance cardiometabolic health and neuromuscular fitness in youth [11–13]. Additional research on the acute responses to INT could be used to establish preliminary cardiometabolic references values for INT and inform the design of novel exercise interventions for children. Therefore, the purpose of this study was to examine the acute cardiometabolic responses to a multi-modal INT protocol in children and to compare these responses to a bout of moderate-intensity treadmill walking. Based on previous findings regarding the acute cardiometabolic responses to different exercise modalities in youth [14,15], we hypothesized that selected INT exercises would elicit a cardiometabolic response that was equal to or greater than brisk walking in children. #### 2. Materials and Methods #### 2.1. Participants A convenience sample of 14 healthy children (8 boys and 6 girls; mean ± SD age 10.7 ± 1.1 years; height 143.2 ± 6.8 cm and body mass 36.3 ± 9.9 kg) volunteered to participate in this study. Participants were active members of local sports teams (primarily soccer and lacrosse), but none participated regularly in resistance training. Parents completed a modified physical activity readiness questionnaire to evaluate the health status of the participants and assess the safety for performing vigorous exercise. All parents signed a parental permission form and all participants signed a child assent form and were informed of the benefits and risks of this investigation. This study was approved by the Institutional Review Board at The College of New Jersey (process No: 2017-0080, 7 February 2018). #### 2.2. Peak Aerobic Capacity Testing All participants reported to the Human Performance Laboratory at least 2 h postprandial for peak aerobic capacity testing. VO2 peak was assessed using the Fitkids treadmill test protocol [24] and a metabolic system (MedGraphics ULTIMA Metabolic System, MedGraphics Corporation, St. Paul, MN, USA). The Fitkids treadmill test is a valid and reproducible exercise test for children that consists of 90 s stages with incremental increases in speed and incline until volitional exhaustion [24]. Breath-by-breath VO2 data were obtained and VO2 peak was determined by recording the highest measure observed during the test [25]. HR was monitored using a soft chest strap with a HR sensor (Model A300; Polar Electro Inc., Woodbury, NY, USA). HR peak was defined as the highest value achieved during the test. Participants were asked to manually signal without verbalizing their rating of perceived exertion (RPE) during the test [26]. Prior to testing, height was measured to the nearest 0.1 cm using a wall-mounted stadiometer and body mass was measured to the nearest 0.5 kg using an electronic scale. For both measurements, participants wore light cloths and no shoes. #### 2.3. Integrative Neuromuscular Training Protocol Participants returned to the Human Performance Laboratory to perform the INT protocol within 2 to 7 days of the peak aerobic capacity test. The INT protocol used in this study was based on previous pediatric research [11,14,15,27] and included strength- and skill-building exercises that were appropriate for children [10]. Our INT protocol consisted of the following six exercises: (1) balance board squats (EX1; 15 repetitions), (2) medicine ball squats with toss and catch (EX2; 15 repetitions), (3) BOSU™ planks with side steps (EX3; 20 repetitions), (4) medicine ball forward lunges (EX4; 16 repetitions), (5) battling rope double arm waves (EX5; 30 repetitions) and medicine balls slams (EX6; 15 repetitions). The 6 INT exercises were performed in successive order with each exercise interval lasting 30 s in duration. Each exercise was performed twice with a rest interval of 30 s in between sets and exercises. The 12 time intervals corresponding to the initiation and termination of each 30 s INT set were carefully monitored and labeled. The total duration of the INT protocol was 12 min (including 30 s recovery following the last exercise). Pilot testing from our center found that a 2.3 kg medicine ball and a 4.1 kg battling rope were appropriate for children. Participants were asked to follow a specific cadence using a metronome and to try and complete a target number of repetitions during each set. A research assistant performed the INT protocol at the desired cadence with each participant during each set and provided a quick review of the upcoming exercise during each rest interval. All participants performed the same exercises in the same order. Participants were asked to manually indicate their RPE after each INT set on a visually presented scale consisting of verbal expressions with a numerical response range of 0 to 10 and five pictorial descriptors that represent a child at varying levels of exertion [26]. Participants became familiar with the INT protocol during a familiarization session which took place after the peak aerobic capacity test. During the familiarization session participants practiced each INT exercise and proper technique was reinforced with exercise-specific coaching cues. #### 2.4. Treadmill Protocol Participants performed the TM trial 2 to 7 days after the INT protocol. The TM protocol used in this study was designed to be a moderate-intensity walking protocol that is consistent with general physical activity recommendations for school-age children [28,29]. Participants walked briskly at a predetermined exercise intensity of 50% VO2 peak for 12 min. The speed and grade of the treadmill were adjusted to maintain the desired exercise intensity throughout the 12-min session. Cardiometabolic data were collected during the same 30 s time intervals as INT. Participants were asked to manually indicate their RPE during the TM trial [26]. #### 2.5. Experimental Measurements: Oxygen Uptake and Heart Rate VO2 and HR were measured at rest and throughout the INT and TM protocols following similar experimental procedures. On arrival, each participant was asked to drink water ad libitum to prehydrate and was fitted with the same child-size respiratory mask and heart rate monitor used for the maximal aerobic capacity test. HR data were downloaded for analysis using a computer software program. HR data analyzed were the mean values collected during each 30-s time interval throughout the 12-min INT and TM protocols. Breath-by-breath VO2 was measured during the INT and TM protocols using the same metabolic system used for maximal aerobic capacity testing. Values for relative VO2, minute ventilation (VE) and respiratory exchange ratio (RER) were recorded during the entire protocol. Individual breath-by-breath data points for all metabolic variables were averaged for each 30 s interval. Prior to each trial, each participant sat quietly in a chair for 5 min to collect baseline data. Once complete, the researcher briefly reviewed session instructions. Subsequently, each participant performed 2 to 3 min of calisthenics (e.g., arm circles and knee lifts) prior to INT or 2 to 3 min of low intensity walking prior to TM. Verbal encouragement was provided throughout the INT and TM trials. #### 2.6. Statistical Analysis Descriptive statistics (mean ± SD) were calculated for all dependent variables. For each protocol, the mean values for VO2, VE, RER, and HR were averaged every 30 s in time-match intervals as well as for the entire 12-min protocol. A 2 (INT or TM) × 12 (time interval sets) analysis of variance with repeated measures was used to analyze within and between participant cardiometabolic and RPE data. A significant F ratio was followed by pairwise comparisons to detect differences between INT and TM at a given time interval using Bonferroni's adjustments. In addition, a dependent t-test was used to compare mean VO2, VE, RER, and HR of the entire protocol between INT and TM. For all statistical tests, a probability level of p < 0.05 denoted statistical significance. Statistical analyses were conducted in SPSS (version 24; SPSS, Chicago, IL, USA). #### 3. Results All participants completed study procedures and no injuries or unexpected events occurred. Our post hoc comparisons revealed a progressive increase in cardiometabolic demand as VO2, VE, RER, and HR increased significantly throughout our multi-exercise INT protocol. During the INT protocol mean HR significantly increased from 121.1 <sup>±</sup> 9.0 b·min−<sup>1</sup> to 183.5 <sup>±</sup> 7.9 b·min−<sup>1</sup> and mean VO2 significantly increased from 14.9 <sup>±</sup> 3.6 mL·kg−1·min−<sup>1</sup> to 34.0 <sup>±</sup> 6.4 mL·kg−1·min−<sup>1</sup> (Table 1). Values for HR, VE, and RER tended to increase with each successive INT exercise and paralleled VO2 data. Mean VO2 and HR during EX5 and EX6 of the INT protocol were significantly higher than during time-matched TM intervals (Table 1) (all Ps < 0.05). Figure 1; Figure 2 depict the gradual increase in HR and VO2, respectively, during the INT protocol as compared to TM. INT. p ≤ 0.05. Figure 1. Heart rate (HR) responses (mean ± SD) during integrative neuromuscular training (INT) and treadmill (TM) protocols. PRE = Baseline; REC = recovery. See Table 1 for significant differences between INT exercises and protocols. Figure 2. Relative oxygen uptake responses (mean ± SD) during integrative neuromuscular training (INT) and treadmill (TM) protocols. PRE = Baseline; See Table 1 for significant differences between INT exercises and protocols. The relative cardiometabolic intensity of each INT exercise (expressed as a percentage of values attained during maximal aerobic capacity testing) ranged from 61.0% to 92.4% for HR and from 28.2% to 64.3% for VO2. The relative cardiometabolic demands of each INT exercise compared to time-matched TM time intervals are outlined on Table 2. The significant increases in cardiometabolic responses during INT mirrored significant increases in RPE. The mean RPEs (out of 10) for INT EX1 to EX6 were 1.14 ± 0.85, 2.26 ± 0.98, 2.91 ± 1.04, 3.55 ± 1.23, 5.42 ± 1.45 and 6.68 ± 1.67, respectively. There was no significant difference in mean VO2 between the entire 12-min INT and TM protocols; however, mean values for HR, VE and RER for the entire 12-min protocol were significantly higher during INT than TM (all Ps < 0.05) (Table 3). The mean VO2 throughout the 12-min TM protocol was 49.5% of VO2 peak attained during maximal aerobic capacity testing. Table 2. Relative cardiometabolic intensity during integrative neuromuscular training (INT) and treadmill (TM) walking intervals. Table 3. Mean cardiometabolic responses during the entire 12 min integrative neuromuscular training (INT) and treadmill (TM) protocols. All values are mean ± SD. \* different than INT. p ≤ 0.05. #### 4. Discussion The aim of our study was to examine the acute cardiometabolic responses to a multi-modal INT protocol in children and to compare these responses to a bout of moderate-intensity TM walking. Consistent with our hypothesis, we found a progressive, multi-modal INT protocol comprising 30 s of work with 30 s of passive recovery can pose a moderate to vigorous cardiometabolic stimulus in children and selected INT exercises can be equal to or more metabolically challenging than TM walking. While other pediatric investigations detailed the acute physiological responses to intermittent bouts of single-mode exercise with cycling, sprinting, medicine balls or battling ropes [14,15,30,31], this is the first study to describe the acute cardiometabolic demands of a mixed-exercise INT protocol in children. No significant differences were observed in mean VO2 between the entire 12-min INT and TM protocols. However, mean HR, VE, and RER were significantly higher during INT than TM. Given the link between vigorous physical activity and positive health outcomes in youth [17–19], our findings provide insight into the potential cardiometabolic benefits of INT if performed at the requisite weekly frequency. Participants in our study were physically active children with a peak aerobic capacity of 52.9 <sup>±</sup> 9.4 mL·kg−1·min−<sup>1</sup> and a peak HR of 198.5 <sup>±</sup> 5.5 bpm. During the INT protocol, VO2 increased from 14.9 <sup>±</sup> 3.6 to 34.0 <sup>±</sup> 6.4 mL·kg−1·min−<sup>1</sup> and HR increased from 121.1 <sup>±</sup> 9.0 bpm to 183.5 ± 7.9 bpm (Table 1). As shown on Table 2, the relative intensity of each INT exercise expressed as a percentage of VO2 peak and HR peak ranged from 28.2% to 64.3% and from 61.0% to 92.4%, respectively. The mean VO2 and HR responses during the entire 12 min INT protocol were 49.5% and 73.1%, respectively, of peak values. When compared to a standard classification of physical activity intensity based on percentage of VO2 peak or HR peak, our findings indicate that the overall intensity of our 12-min INT protocol could be characterized as "moderate" (i.e., 46–63% VO2 peak and 64–76% HR peak) whereas the intensity of individual INT exercises could be characterized as "light", "moderate", or "vigorous" depending upon the mode and complexity of each movement [32]. Knowing the intensity levels of different INT exercises can help researchers and practitioners design interventions that optimize training-induced adaptations and encourage compliance in all participants. The progressive increase in HR throughout our INT protocol was consistent with other reports that examined the acute physiological responses to different modes of resistance exercise in youth [14,15,20]. During a 10-min bout of medicine ball interval training (30 s/exercise and 30 s rest/set) researchers reported that mean HR values during work sets ranged from 121.5 ± 12.3 bpm (61.1% HR peak) to 178.3 ± 9.4 bpm (89.6% HR peak) [14]. Harris and colleagues characterized the acute responses to resistance exercise and HIIT in early adolescents (12–13 years) and reported mean HR over all 12 work sets of 169.9 ± 9.2 bpm for resistance training and 179.0 ± 5.6 bpm for HIIT which represented 85% and 90% of HR peak, respectively [20]. In our study, the gradual increase in HR from 61% HR peak to over 90% HR peak was expected because the structure of our multi-modal INT protocol included six different exercises that progressed from a less intense squatting exercise to a more explosive movement. These findings are notable because high-intensity interval training characterized by short bouts of vigorous intensity activity may be needed to elicit the greatest improvements in cardiometabolic health and aerobic fitness in children [21,33]. Of interest, participants in our study recovered quickly from the demands of INT as evidenced by heart rates of 139.1 ± 13.9 bpm, 118.4 ± 14.2 bpm, and 113.9 ± 13.5 bpm after 1, 3 and 5 min of recovery, respectively (Figure 2). These observations are consistent with others who reported a faster post-exercise recovery heart rate in children than endurance adult athletes. [34]. The findings related to relative VO2, VE and RER build upon previous reports investigating different modes of youth resistance training. During a progressive interval protocol with 5 battling rope exercises, relative VO2, VE and RER increased to 30.0 mL·kg−1·min−<sup>1</sup> (64.8% VO2 peak), 40.8 L/min and 1.07, respectively [15]. In another report, relative VO2, VE and RER reached 34.9 mL·kg−1·min−<sup>1</sup> (63.6% VO2 peak), 40.4 L/min and 0.95, respectively, during medicine ball interval training [14]. While Baquet and colleagues reported mean VO2 values of 35.5 mL·kg−1·min−<sup>1</sup> (64.6% VO2 peak) to 47.0 mL·kg−1·min−<sup>1</sup> (85.9% VO2 peak) during sprint high intensity interval exercise in children [31], differences in exercise characteristics, workload duration and rest interval length can explain, at least in part, these findings. The progressive increase in RER that reached values above 1.0 which were greater than those observed during TM walking further attested to the intense nature of our INT protocol. Collectively, it appears that INT and other types of interval training could be used to bring about positive cardiometabolic adaptations in children. Our findings demonstrate that INT characterized by short exercise intervals interspersed with brief rest intervals can pose a moderate to vigorous cardiometabolic stimulus in youth. Unlike brisk walking, INT typically requires the whole body to function as a unit in order to perform movements proficiently with proper technique at the desired cadence. During our multi-modal INT protocol, the highest VO2 and HR values were achieved during EX5 (battling rope double arm wave) and EX6 (medicine ball slams), respectively. Both of these exercises require a substantial involvement of the upper and lower body since participants vigorously waved a 4.1 kg battling rope with both arms or forcibly slammed a 2.3 kg medicine ball against the floor at maximal or near maximal velocity. Interestingly, Ratamess and colleagues examined the acute cardiometabolic response to 13 different resistance exercise protocols in adults and found that the battling rope double arm wave elicited the highest responses [35]. While the greater complexity and muscle mass activation of the INT exercises towards the end of our protocol suggest that the choice of exercise is a primary determinant of the cardiometabolic responses to INT, the cumulative effects of fatigue and cardiovascular drift during our INT protocol should also be considered because the cardiometabolic responses to each INT exercise were likely influenced by the subsequent fatigue from the previous exercise. The structure of our INT protocol was based on previous fitness interventions with children and included 2 sets of 6 different exercises with a 30 s rest interval in between sets and exercises [11,27]. Due to the age of the participants and the relative intensity of selected INT exercises, a progressive, multi-modal INT protocol with passive recovery intervals was arguably required to maintain safety, motivation and adherence. Sustained bouts (>10 min) of physical activity are rare in children and the natural tempo of their activity is characterized by short bursts lasting a few seconds [36]. Furthermore, it is important to note that the objective of our investigation was not to disentangle the acute demands of each INT exercise, but rather to examine the acute cardiometabolic responses to a novel INT protocol and to compare these responses to a more traditional form of continuous physical activity of comparable time and overall VO2. Notably, we used a time period of 12 min so that the protocol could be readily incorporated into a physical education class or sports practice. With that said, the results from our investigation provide preliminary cardiometabolic reference values for a multi-modal INT protocol and highlight the versatility of INT because different exercises could be combined to offer light, moderate, vigorous or variable intensity training depending on the needs, goals and abilities of the children. Our findings support the integration of INT into school- and community-based youth fitness programs and inform the development of interventions aimed at increasing time spent in MVPA. The progressive increase in cardiometabolic intensity throughout the INT protocol was consistent with the participants perceptions as evidenced by significant increases in RPE. Others found that youth could rate their perceived exertion during resistance training and our observations support the use of RPE to monitor the intensity of INT in children [20,37]. We also observed that our INT protocol was challenging and appealing for the participants. This was evidenced by 100% compliance with research instructions and testing protocols. Although the affective response to INT were not explored in our investigation, Malik and colleagues found that enjoyment was higher following high-intensity interval exercise compared with continuous moderate-intensity exercise in adolescents [38]. Given that a child's level of enjoyment is a strong predictor of physical activity participation [39], further examination of the affective responses to INT are warranted. The INT exercises used in our investigation were intended to progress from less intense to more intense. EX1 consisted of squatting on a balance board at a controlled cadence and EX6 required participants to repeatedly slam a 2.3 kg medicine ball against the floor with energy and vigor. These are important considerations when discussing our findings because the cardiometabolic responses to INT are dependent upon various factors including the intensity of muscle actions, type of muscle actions, the amount of muscle mass used, rest intervals and body position [14,15,40]. In addition, age, fitness level and body mass index can influence the acute cardiometabolic responses to exercise and the kinetics of recovery after exercise [25,34,41]. Children appear to be less susceptible to neuromuscular fatigue than adults following resistance training [42] and the post-exercise decline in VO2 seems to be faster in children with a higher peak VO2 than those with a lower peak VO2 [34,43]. Thus, the results of our investigation should be interpreted within the context of a mixed model exercise approach that reflects how children may actually perform INT during physical education or sports practice. We acknowledge that the maturity status of the participants was not assessed and therefore we were unable to determine if all participants were prepubertal. Also, the participants in our study were active, healthy girls and boys so the homogeneity of our sample limits generalizability to other populations including those with illnesses or disabilities that alter movement or mechanical efficiency. It is also important to consider the design of our INT protocol and the limited INT experience of our participants. Acute program variables will impact the cardiometabolic responses to INT and as children become more skilled and efficient at performing INT exercises the acute cardiometabolic demands to a given protocol will not be constant. While other pediatric researchers examined the acute cardiometabolic responses to single-mode exercise, the design of our progressive, multi-modal INT protocol arguably provides greater translatability to school- and community-based programs in which the physical fitness levels and activity interests of children can vary widely. #### 5. Conclusions INT has been investigated as a potentially potent and time efficient method of enhancing neuromuscular fitness in youth [11,12,16] and our novel findings suggest that this innovative training method consisting of strength- and skill-building exercises could also provide a sufficient training stimulus needed to induce cardiometabolic adaptations. Considering the amount of time children spend in MVPA during physical education and youth sport practice is falling short of expectations [44,45], INT could be a worthwhile addition to school- and community-based programs to target exercise deficits. Unlike continuous bouts of moderate-intensity walking, a progressive INT intervention with battling rope and medicine ball exercises may better prepare youth for the vigorous intensity nature of game and sport activities. These observations have practical relevance for teachers, coaches and health care providers who design exercise programs and sport practices for children. Altogether, the acute cardiometabolic responses to INT along with high compliance to our study procedures provide support for future training studies to better understand the multidimensional benefits of INT on health, fitness and performance in youth. Author Contributions: Conceptualization, A.D.F., J.K. and N.A.R.; Formal analysis, A.D.F., J.K., N.A.R. and J.B.; Investigation, A.D.F., J.K., A.C.F., M.B., S.D. and C.J.; Methodology, A.D.F., J.K., M.B., S.D. and C.J.; Resources, A.D.F., J.K., N.A.R., A.C.F. and J.B.; Supervision, A.D.F., J.K. and A.C.F.; Writing—original draft, A.D.F. and J.K.; Writing—review & editing, A.D.F., J.K., N.A.R., A.C.F., M.B., S.D., C.J. and J.B. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).	doab	2025-04-07T03:56:58.859823	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 11 }
0051d55d-461b-414c-a51b-301e45ff4d0c.12	Article Validation of Cardiorespiratory Fitness Measurements in Adolescents Pedro Migliano <sup>1</sup> , Laura S. Kabiri <sup>2</sup> , Megan Cross 1, Allison Butcher <sup>1</sup> , Amy Frugé 1, Wayne Brewer <sup>1</sup> and Alexis Ortiz 3,\* Received: 28 June 2019; Accepted: 11 July 2019; Published: 13 July 2019 Abstract: Cardiorespiratory fitness (CRF) is an important indicator of adolescent cardiovascular well-being and future cardiometabolic health but not always feasible to measure. The purpose of this study was to estimate the concurrent validity of the non-exercise test (NET) for adolescents against the Progressive Aerobic Capacity Endurance Run (PACER®) and direct measures of VO2max as well as to examine the concurrent validity of the PACER® with a portable metabolic system (K4b2™). Forty-six adolescents (12–17 years) completed the NET prior to performing the PACER® while wearing the K4b2™. The obtained VO2max values were compared using linear regression, intra-class correlation (ICC), and Bland–Altman plots, and α was set at 0.05. The VO2max acquired directly from the K4b2™ was significantly correlated to the VO2max indirectly estimated from the NET (r = 0.73, p < 0.001, r<sup>2</sup> = 0.53, ICC = 0.67). PACER® results were significantly related to the VO2max estimates from the NET (r = 0.81, p < 0.001, r<sup>2</sup> = 0.65, ICC = 0.72). Direct measures from the K4b2™ were significantly correlated to the VO2max estimates from the PACER® (r = 0.87, p < 0.001, r<sup>2</sup> = 0.75, ICC = 0.93). The NET is a valid measure of CRF in adolescents and can be used when an exercise test is not feasible. Keywords: VO2max; PACER; non-exercise test #### 1. Introduction Low cardiorespiratory fitness (CRF) levels in adolescents have been linked to insulin resistance as well as cardiovascular and cardiometabolic risk factors [1–5]. Higher levels of CRF in adolescents have also been linked to improved academic outcomes and an improved ability to regulate attention and behavior [6,7]. There is a strong positive relationship between CRF and cognitive performance, with multiple studies showing that adolescents with higher levels of CRF outperformed their peers with lower levels of CRF in cognitive tests involving working memory, cognitive flexibility, and inhibition, which is the ability to ignore extraneous environmental information [8–10]. The improved cognitive ability is thought to be due to the increased volume in different regions of the brain such as the hippocampus and basal ganglia found in adolescents with higher levels of CRF [8,11]. Moreover, higher CRF has been shown to be an excellent predictor of physical activity level during adulthood [12,13]. Since CRF is an important health predictor in adolescents, it should be regularly assessed in both healthcare and school settings. Maximal oxygen uptake (VO2max), expressed as mL/kg/min of oxygen, is the gold standard measure of CRF and can be measured both directly and indirectly [14,15]. A direct measure of VO2max is obtained by ventilatory gas analysis during a graded exercise test at maximum exertion and is the most precise measure of VO2max [14]. An indirect measure estimates VO2max from a maximal or submaximal exercise test and is usually estimated from total time, total work, or heart rate [15]. Especially in school settings, VO2max is often indirectly measured and estimated using a field test. While there are a few different options when it comes to field tests to indirectly measure VO2max, the 20-m multistage shuttle run test (20MSR) is the most frequently used test, as it has shown moderate to high criterion validity compared to direct measures of VO2max and is a more practical test compared to longer duration endurance tests, such as the 6-min endurance run [16,17]. The Progressive Aerobic Cardiovascular Endurance Run (PACER®) is part of the FitnessGram® tests administered to adolescents in schools [18]. The PACER® is a 20MSR meant to mimic a maximal exertion exercise test with workloads increasing every stage until the participant reaches volitional exhaustion. The standardized procedure of the PACER® has participants run between two markers placed 20 m apart while keeping pace to a prerecorded audio cadence that increases every minute. The test is terminated if a participant fails to reach a marker for the second time or if the participant can no longer continue. This field test has been shown to be both reliable (intra-class correlation (ICC): 0.77–0.93) and valid (r: 0.62–0.83) [16,19,20], when compared to directly measured VO2max during a treadmill maximal exercise test. It is not always possible to directly assess VO2max in all settings, such as schools, gyms, or even in a clinical setting. Ventilatory gas analysis is time-consuming and requires expensive equipment that prevents the mass screening which would be required in a school setting or in the daily caseload and time restraints of a healthcare professional. The non-exercise test (NET) is a paper-based questionnaire used to estimate VO2max from different variables including sex, age, body mass index (BMI), resting heart rate, and self-reported habitual physical activity levels [21,22]. Use of this questionnaire allows for both quick and widespread CRF screening without any health risk to the participants. The NET has been shown to be valid within the adult population [21], but to the best of our knowledge, has not been tested within the adolescent population. Therefore, the purpose of this study was to estimate the concurrent validity of the NET for adolescents against the PACER® test and objective measures of VO2max as well as to examine the concurrent validity of the VO2max estimated by the PACER® with the VO2max obtained from the Cosmed K4b2™ portable metabolic system (Cosmed K4b2, Cosmed, Rome, Italy) during the PACER® test. #### 2. Materials and Methods #### 2.1. Subjects Adolescents aged 12–17 years were recruited by email, education support groups, co-operatives, and word of mouth. Parents were asked if their child had any physical or mental limitations that would prevent them from safely and accurately completing the test and if any were noted, the child was excluded. Institutional review board approval (Protocol #19736 on 19 January 2017) from Texas Woman's University in Houston, TX, parental informed consent, and minor assent were secured prior to any subject enrollment or data collection. #### 2.2. Procedures Height and weight were assessed barefoot with light clothing using a medical-grade stadiometer calibrated prior to its use. Pubertal level was assessed using the pubertal developmental scale. Prior to participating in the PACER and being fitted with the K4b2™, participants were given the NET questionnaire previously validated in adults by Jurca et al. [21]. As required by the NET, each participant was asked to choose the physical activity level which best described their daily level of activity, as listed in Table 1. Participant BMI was calculated based on their weight and height measurement. Resting heart rate (RHR) was measured with the SantaMedical SM150BL finger pulse oximeter (SantaMedical, Tustin, CA, USA) as the final measurement after sitting for at least five minutes. NET equation: VO2max = 3.5 \* ((Sex \* 2.77) − (age \* 0.1) − (BMI\0.17) − (RHR \ 0.03) + (physical activity score \* 1.00) + 18.07); Sex: 0 = F, 1 = M. The K4b2™ portable metabolic system was used to directly measure VO2max during the PACER®. The K4b2™ has demonstrated high test–retest reliability (ICC = 0.70–0.90) [23] and has shown to be valid in VO2 measurement when compared to a traditional stationary gas exchange system (r: 0.93–0.97) [24]. The K4b2™ was calibrated according to manufacturer's recommendations using known gases and room air sampling. After calibration, participants were fitted with a mask and connected to the K4b2™ prior to initiating the PACER®. After donning the K4b2™, adolescents were allowed to wear and familiarize themselves with the device for 5 min. Participants completed the 20-m PACER® individually as per the standardized instructions. Two pieces of tape were placed 20 m apart, denoting the crossing lines for the PACER®, and the FitnessGram® cadence soundtrack was played at a volume loud enough for the participant to hear. Participants were instructed to begin running when they heard the first beep on the soundtrack. For the lap to be valid, each participant was required to clear at least one foot over the line prior to the next beep. The test was terminated following the second miss or if the participant desired to stop. All raters were trained and followed the standardized test procedures. Once the PACER® had been terminated, total laps were recorded, the K4b2™ data was saved, and the mask was doffed. VO2max with the K4b2™ system was determined in the same manner used by Silva et al. [25]. Respiratory variables were recorded breath-by-breath and averaged over a 10-s period. VO2max was determined when a plateau in the VO2 curve was detected and if the plateau was absent the VO2peak was taken instead. VO2max with the PACER® was calculated using the quadratic model established by Mahar, Guerieri, Hanna, and Kemble [26]: VO2max <sup>=</sup> 41.76799 <sup>+</sup> (0.49261 <sup>×</sup> laps) <sup>−</sup> (0.00290 x laps2) <sup>−</sup> (0.61613 × BMI) + (0.34787 × sex × age), where sex is 0 for males and 1 for females. #### 2.3. Statistical Analyses The Shapiro–Wilk test, Levene's test, and box plots were utilized to screen all data for normality assumptions, homoscedasticity, and outliers, respectively. Linear regressions with Pearson correlations were performed on the VO2max estimates acquired from the PACER® and the NET, and the VO2max acquired from the K4b2™ system. Intra-class correlations (ICCs), standard error of estimate (SEE), paired sample t-tests, and Bland–Altman plots were performed to assess agreement and differences between the measures of VO2max. All statistical analyses were conducted with IBM SPSS software for Windows (v. 25.0; IBM Corp., Armonk, NY, USA). #### 3. Results Forty-six adolescents (male: 24; female: 22) were recruited. All data met all assumptions for normality, homoscedasticity, and outlier assumptions. Sex specific demographic and anthropometric characteristics are listed in Table 2. Table 2. Demographic and anthropometric characteristics. Pearson correlation coefficients as well as ICC and SEE values are listed in Table 3. Scatterplots depicting the correlations are listed in Figure 1. All assessment methods demonstrated moderate to strong statistically significant correlations and levels of agreement as defined by Portney and Watkins [27] for correlations and Koo and Lee [28] for ICC. Table 3. Correlation matrix between oxygen consumption estimates per test for both sexes combined. Figure 1. Cont. A Figure 1. Simple scatterplots. (A) NET & K4b2: r = 0.73; (B) NET & PACER®: r = 0.81; (C) K4b2 & PACER®: r = 0.87; Bland–Altman plots (Figure 2) and paired sample t-tests demonstrated acceptable limits of agreement and no significant difference for K4b2™ and PACER® measures of VO2max (Figure 2A) (mean difference = −0.37, t(45) = −0.61, p = 0.55) but showed that the NET VO2max estimates tend to be overestimated, seen by the upward shift of the mean difference line away from zero, and significantly different in the NET <sup>−</sup> PACER® (Figure 2B) (mean difference <sup>=</sup> 7.14, <sup>t</sup>(45) <sup>=</sup> 10.26, <sup>p</sup> <sup>&</sup>lt; 0.001) and the NET <sup>−</sup> K4b2™ (Figure 2C) (mean difference <sup>=</sup> 7.52, <sup>t</sup>(45) <sup>=</sup> 8.54, <sup>p</sup> <sup>&</sup>lt; 0.001) plots. Figure 2. Cont. Figure 2. Bland–Altman plots. Middle line represents mean difference while other dotted lines represent the limits of agreement: <sup>±</sup>1.96s. (A) K4b<sup>2</sup> & PACER®: mean difference <sup>=</sup> <sup>−</sup>0.37, limits of agreement of <sup>−</sup>8.61 & 7.85; (B) NET & PACER®: mean difference <sup>=</sup> 7.14, limits of agreement of <sup>−</sup>2.10 & 16.39; (C) K4b2 & NET: mean difference <sup>=</sup> 7.52, limits of agreement of <sup>−</sup>4.19 & 19.23. #### 4. Discussion The aim of this study was to estimate the concurrent validity of the NET for adolescents against the PACER® and objective measures of VO2max as well as to examine the concurrent validity of the VO2max estimation by the PACER® with the VO2max from the K4b2™ portable metabolic system during the PACER® test. Past studies have shown that the NET is a valid measure of CRF in adult populations [21,22], and our results show that the NET is a valid predictor of VO2max in the adolescent population. This is important, as it offers a quick, easy, inexpensive, and risk-free method of assessing CRF in adolescents. To our knowledge, this is the first report to measure CRF in adolescents without requiring any form of exertion from the participant, which allows for widespread use among large groups and offers a feasible tool for CRF measurement in healthcare settings. The PACER® test has been validated several times [16,19,20], and our results comparing it to the direct measure of VO2max from the K4b2™ (r = 0.87) align with past results (r = 0.62–0.83) acquired directly from treadmill maximal exercise tests [16,19,20]. Our study offers a different perspective, as most studies compare the PACER® estimate to a traditional treadmill maximal exercise test, but our research compared the estimate to the direct measure of VO2max acquired at the same time the participant was performing the PACER®. This further strengthens the validity of the PACER® as an indirect measure of VO2max and shows that the PACER® protocol is a valid substitute for a maximally graded exercise test. In practice, the NET could offer schools a quick and inexpensive tool to assess CRF more frequently than the PACER® test allows, providing valuable information to implement changes in physical education. At the clinical level, the NET can be implemented into healthcare settings as a snapshot of current CRF and can be used as a tool to assess risk for cardiovascular and cardiometabolic disease [1–5]. For efficient use in either setting, the NET results can be analyzed similar to the PACER®, which groups students based on having sufficient or insufficient CRF into the healthy fitness zone (HFZ), the needs improvement zone (NIZ), or the health risk zone (HRZ) [18]. Based on our results, the group values for the zones could be adjusted with the linear regression equation: PACER® = 10.92 + 0.96(NET). For example, a 10-year-old male with a NET score of 46.82–49.42 would fall into the NIZ, a score of ≤46.81 would put him in the HRZ, and a score ≥49.43 would place him into the HFZ. This would allow for a quick assessment to deduce whether an adolescent patient is at risk and requires intervention. As seen with the Bland–Altman plots and t-tests, the NET VO2max estimates were generally much higher and were significantly different than the values acquired directly through the K4b2™ or estimated through the PACER®. This could be due to the fact the descriptions of the physical activity levels were intended for adults, so it may not have been directly relatable to adolescents. Also, the added weight of the K4b2™ unit, weighing roughly 1 kg, plus the addition of the mask could have resulted in a decreased outcome during the PACER® test. While the NET measures of VO2max were significantly higher, the moderately strong levels of agreement show that the NET is valid and by using the CRF grouping suggested above, as is used by the PACER®, the NET overestimation is inconsequential. Limitations of this study include a small sample of females and males, thus limiting the ability to fully explore sexes separately, and the running of the PACER® test individually instead of in groups, as is the norm in schools, thus potentially affecting participants' motivation to maximally exert themselves. Regardless of the limitations of this study, there are several strengths in our design and findings, including validating the NET against two different validated measures of VO2max and testing a representative sample of adolescents. Future studies on using the NET for adolescents could examine a larger sample size, perhaps in public schools, to compare against the PACER® or investigate the NET's sensitivity to change in an adolescent's CRF. Future studies need to also assess the reliability of the NET in adolescents, so it can be established as a tool to measure CRF over time. #### 5. Conclusions This article gives researchers, clinicians, and coaches a simple, quick, and inexpensive tool to indirectly estimate VO2max in the adolescent population as opposed to a time-consuming and exhausting exercise test. This article also strengthens the evidence for use of the PACER® test as an indirect measure of VO2max, as it provided ventilatory gas analysis during the test itself, showing that the PACER® is a valid graded maximal exertion exercise test, as direct measures of VO2max acquired during performing the PACER® were in agreement with PACER® estimates based on regression equations established against the more traditional treadmill VO2max tests. Author Contributions: Conceptualization: A.O., L.K., P.M.; Methodology: A.O.; Formal Analysis: P.M.; Investigation: P.M., M.C., A.B., A.F. L.K., A.O., W.B.; Resources: L.K., A.O., W.B.; Data Curation: P.M., M.C.; Writing—Original Draft Preparation: P.M.; Writing—Review and Editing: P.M., A.O., L.K.; Supervision: A.O., L.K., W.B. Funding: This study was funded by the Texas Physical Therapy Foundation. The results of this study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results from this study do not constitute endorsement by the Texas Physical Therapy Association. Conflicts of Interest: The authors have no conflict of interest to disclose. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). #### Article	doab	2025-04-07T03:56:58.862007	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 12 }
0051d55d-461b-414c-a51b-301e45ff4d0c.13	Anthropometric Obesity Indices, Body Fat Percentage, and Grip Strength in Young Adults with different Physical Activity Levels *Mustafa Sö ˘güt <sup>1</sup> , Ömer Barı¸s Kaya 1, Kübra Altunsoy 1, Cain C. T. Clark 2,\ , Filipe Manuel Clemente 3,4 and Ali Ahmet Do ˘gan <sup>1</sup> Received: 5 July 2019; Accepted: 30 July 2019; Published: 31 July 2019 Abstract:** The purposes of this study were to determine whether moderately physically active (MPA) and highly physically active (HPA) male (n = 96, age = 22.5 ± 1.7 years) and female (n = 85, age = 21.3 ± 1.6 years) young adults differed in their anthropometric obesity indices (AOIs), body fat percentage (BF%), and muscular strength, and also to examine the associations between physical activity level (PAL) and the abovementioned variables. Participants were measured for body height and weight, BF%, waist and hip circumferences, and maximal isometric grip strength. According to their PAL, estimated by the short version of the International Physical Activity Questionnaire, they were assigned to MPA and HPA subgroups. Regardless of gender, results indicated that participants in the MPA groups had significantly higher values of body weight, waist and hip circumference, BF%, and BMI than participants in the HPA groups. No significant differences were found between physical activity groups in terms of grip strength. The AOIs and BF% were found to be significantly and negatively correlated with the PAL in both genders. In conclusion, the findings of the study suggest that high habitual physical activity is associated with lower adiposity markers. However, the differences in the hand grip strength of the contrasting activity groups were negligible. Keywords: obesity indices; body composition; grip strength; physical activity #### 1. Introduction The consequences of physical inactivity, including a diverse range of chronic and non-communicable diseases is well-documented, such as coronary heart disease [1], breast and colon cancers [2,3], osteoporosis [4], noninsulin-dependent diabetes [5], excess adiposity [6], depression [7], and all-cause mortality [8]. Nevertheless, sedentary lifestyle behaviors are still common worldwide [9], and it presents a major global public health concern. Accumulating evidence suggests that habitual physical activity has a positive influence on various health outcomes [10–12]. The findings of large, recent cohort studies have indicated that physical activity level (PAL) is inversely associated with body fat percentage (BF%), body mass index (BMI), and/or other adiposity indicators [13–16]. Notwithstanding, inconsistent results have been observed in certain age groups with regard to the relationship between physical activity and muscular strength [17–20]. Muscular strength is a determinant of physical quality that ensures a lower cardiovascular risk factor [21], a healthy mineral bone density, and high levels of lean mass [22], independently of other fitness variables or sociodemographic measures. Despite there being many ways to assess muscular strength, one of the most common tests carried out in adults is grip strength, which is an important clinical and prognostic value [23]. Usually, strong and positive correlations have been found between hand grip strength and muscle mass [24]. On the other hand, low hand grip strength is associated with increased risk of functional limitations and disability in one's later years, as well as a general cause of mortality [25]. In spite of such evidence, there is a lack of studies testing the relationship between PALs and strength, and this should be considered to understand the mechanism that explains good maintenance of strength. Although young adulthood is a critical developmental period characterized by important changes in health status, such as unhealthy weight gain [26,27] and physical inactivity [28], the majority of the relevant examinations have been conducted with children and older adults. Thus, current literature provides limited evidence on the disparities in adiposity and muscular strength in university students with different PALs. Therefore, the purposes of the present study were to examine whether moderately physically active (MPA) and highly physically active (HPA) male and female young adults differed in their anthropometric obesity indices (AOIs), BF%, and muscular strength, and to analyze the relations between PAL and the aforementioned variables. #### 2. Materials and Methods #### 2.1. Participants A cohort of male (n = 96, age = 22.5 ± 1.7 years) and female (n = 85, age = 21.3 ± 1.6 years) university students from a state university located in central Anatolia (Turkey) were recruited to take part in the study. They were initially briefed of the measurement procedures and the aims of the study, and then requested to sign informed consent forms. Ethical approval was obtained from the Non-interventional Researches Ethics Board of Kırıkkale University (approval number/identification code of the study is 2019.06.25). #### 2.2. Anthropometric Measurements Anthropometric assessments were performed in accordance with standardized procedures [29]. Body height was measured with a portable stadiometer (Seca 213, Hamburg, Germany) to the nearest 0.1 cm. Body mass (0.1 kg) and BF% were evaluated by a bioelectrical impedance analyzer (Tanita, BC-418, Japan). Waist and hip circumferences were measured with a flexible steel tape to the nearest centimeter at the smallest circumference between the ribs and the iliac crest and at the level of maximum protuberance of the buttocks, respectively. BMI was calculated by dividing the body weight (kg) by the body height squared (m). #### 2.3. Grip Strength A digital hand dynamometer (T.K.K.5401 Grip-D, Takei, Japan) was used to assess maximal isometric grip strength. Participants were asked to stay in a standing position and keep one arm straight and parallel to the body. They were requested to hold the dynamometer with their dominant hand and squeeze the handle as hard as possible for three seconds. The highest value acquired from the three trials was used for the analysis. #### 2.4. Physical Activity The PAL of the participants was estimated via the short form of the International Physical Activity Questionnaire (IPAQ) [30,31]. The IPAQ requires respondents to recall the frequency, duration, and intensity of physical activity they engaged in during the last seven days. Each type of activity (moderateand vigorous-intensity walking) was then converted to metabolic equivalent (MET) minutes, enabling the determination of total weekly physical activity. Based on their weekly MET minutes, they were divided into two groups—MPA (≥600–3000 MET min/week) and HPA (≥3000 MET min/week) [30,31]. #### 2.5. Statistical Analysis Data were analyzed using SPSS for Windows. Descriptive statistics (mean ± SD) were calculated for all variables. An independent sample t-test was used to ascertain differences between PAL groups. Effect sizes (ES) were quantified to determine the magnitude of differences. Based on the Cohen's d values, ES were considered as: <0.20 (trivial), 0.20 to 0.59 (small), 0.60 to 1.19 (moderate), 1.20 to 1.99 (large), 2.0 to 3.9 (very large), and >4.0 (extremely large) [32]. A Pearson correlation coefficient was conducted to examine the associations between PAL and other variables. Correlations were categorized as 0.0–0.1 (trivial), 0.1–0.3 (small), 0.3–0.5 (moderate), 0.5–0.7 (large), 0.7–0.9 (very large), and 0.9–1.0 (near perfect) [32]. #### 3. Results Descriptive statistics (mean ± SD), t-test results, and effect sizes for male and female participants are presented in Tables 1 and 2. Results highlighted that participants in the HPA groups had significantly lower values in BF%, body mass, waist and hip circumference, and BMI than the participants in the MPA groups in both genders. There were no significant differences in age, body height, and grip strength results between groups. Table 1. Descriptive statistics, t-test results, and effect size values for male participants. Table 2. Descriptive statistics, t-test results, and effect size values for female participants. Table 3 presents the correlation coefficients between PAL and other variables for each gender, separately. Results indicated that the PAL, regardless of gender, was found to be significantly and negatively correlated with all AOIs and BF%. On the other hand, no significant associations were found between PAL and grip strength values in both male and female participants. Table 3. Correlation results between physical activity level (PAL) and other variables by gender. #### 4. Discussion The aim of the present study was to investigate the possible differences in various adiposity parameters and muscular strength in a group of young male and female adults with distinct physical activity profiles. The results of the study indicated that the participants in the HPA group had significantly lower values in all AOIs and BF% than their MPA counterparts in both genders. Furthermore, regardless of gender, PAL was found to be significantly and negatively correlated with AOIs and BF%. The results, with regard to reported correlation coefficients, are in line with the findings of previous longitudinal [33–35] and cross-sectional [36,37] examinations. Concordant with our findings, results of some recent work noted similar observations, that individuals who engaged in greater physical activity were found to have preferential indices of adiposity [13,14]. The results demonstrated that there were no significant differences in grip strength results between activity groups. This observation is in accord with the findings of previous studies conducted with children [38], young adults [39], and older adults [40]. Furthermore, no significant associations were obtained between PAL and grip strength values in both genders. This might conceivably be attributed to comparable body sizes, where trivial and small magnitudes of differences were found between groups for body height and weight, respectively. Furthermore, the participants were grouped by the IPAQ, which does not specifically assess participation in strengthening activities, so may not be sufficiently sensitive. In addition, other possible anthropometric correlates of hand grip strength that were not measured in this study, such as hand size and forearm circumference [41], total arm length and upper arm circumference [42], and hand circumference [43] might contribute to this insignificance, and therefore warrants further examination. With regard to practical implications, it is conceivable that high PALs and good levels of strength may help to maintain health levels across adulthood. Different studies have suggested that upper and lower body muscular strength are important factors that contribute to a lower risk of mortality in the adult population, regardless of age [44,45]. Although no differences were found between PALs in strength, it is important to highlight that strength is an independent physical quality and should be worked out concurrently with other fitness components (e.g., cardiorespiratory fitness). Thus, a healthy lifestyle should include both cardiorespiratory and muscular strength training/stimuli [46]. #### 5. Conclusions The findings of the present study revealed that higher levels of habitual physical activity is associated with lower adiposity markers in young adults. Nevertheless, differences in hand grip strength of the contrasting activity groups were negligible. It should also be acknowledged that the current study has several limitations. Firstly, the subjective assessment may cause incorrect estimation of physical activity; however, use of the well-validated IPAQ ameliorates some concern. Secondly, in spite of its important advantages (i.e., being relatively inexpensive, portable, and quick), rather than BIA, criterion methods such as underwater weighing or dual-energy x-ray absorptiometry might be used to determine body fat, but nevertheless present significant time and cost restrictions. Nevertheless, it is recommended that future studies expand this observation through using a larger sample and more complex reference methods to measure physical activity levels and body composition. Author Contributions: Conceptualization, M.S. and O.B.K.; Data curation, M.S. and O.B.K.; Formal analysis, M.S.; Investigation, K.A. and F.M.C.; Software, M.S.; Supervision, C.C.T.C. and A.A.D.; Writing—original draft, M.S., O.B.K., K.A., C.C.T.C. and F.M.C.; Writing—review and editing, C.C.T.C. and A.A.D. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). #### Article	doab	2025-04-07T03:56:58.863217	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 13 }
0051d55d-461b-414c-a51b-301e45ff4d0c.14	Accelerometer-Based Physical Activity Levels Differ between Week and Weekend Days in British Preschool Children #### *Clare M. P. Roscoe 1,\ , Rob S. James <sup>2</sup> and Michael J. Duncan <sup>2</sup> Received: 10 August 2019; Accepted: 7 September 2019; Published: 12 September 2019 Abstract:** Participation in physical activity (PA) is fundamental to children's future health. Studies examining the temporal pattern of PA between weekdays and weekends in British preschool children are lacking. Therefore, the aim of this study was to compare PA levels between week and weekend days for UK preschool children, using objective measurements. One hundred and eighty-five preschool children (99 boys, 86 girls, aged 4–5 years), from central England wore a triaxial accelerometer (GENEActiv) for 4 days to determine PA. The time (min) and percentage (%) of time spent in light, moderate and vigorous PA (MVPA) was determined using specific cut-points for counts per minute related to 3–5 year olds. Of the sample, none of the children met the UK recommended 180 min or more of PA per day. A significant difference (P < 0.05) was observed between the amount of time that preschool children spent in sedentary behaviours on weekdays (91.9%) compared to weekend days (96.9%). During weekdays and weekend days, 6.3% and 2.0% of time was spent in MVPA, respectively. Therefore, a substantial proportion of British preschool children's day is spent in sedentary behaviours, with less MVPA accrued during the weekend. Regular engagement during the weekdays provides opportunities to accrue PA, which may not be present on weekend days. Keywords: physical activity; preschool children; health promotion #### 1. Introduction Physical activity (PA) during preschool years is critical to a child's development and overall health and well-being [1,2]; therefore, it is important to integrate PA into early childhood [3,4]. In 2016, over 41 million children worldwide under the age of 5 years were estimated to be overweight [5]. Childhood obesity is an increasing public health concern [6] and weight gained by the age of 5 years has been reported as a predictor of being overweight in adulthood [7]. Physical activity levels and sedentary behaviours of children in the UK have been viewed as 'obesogenic' [8,9], with habitual PA declining over recent years and sedentary behaviour being the dominant state of children's PA levels during their preschool day [4,10–12]. Although studies have examined PA in children aged 5 years and above, fewer studies have been conducted with preschool children. This limited evidence base in UK preschool children's PA levels is therefore a cause for concern. It has been recommended that preschool children in the UK should ideally be participating in at least 180 min of PA per day [13–15]. Studies have discovered that preschool children spend the majority of their day in sedentary behaviours and a low proportion of their day in moderate to vigorous PA (MVPA) (<15%) [10,16,17]. Of children aged between 2 and 4 years in England, only about one in 10 meet the recommendations of at least 180 min of PA per day [18,19]. It has been reported that children engaged in 7.7 min of MVPA per hour at preschool [20]. Therefore, in accordance with Pate et al. [20], if a child, for example, attends preschool for 8 h, they would only engage in ~1 h of MVPA and it is unlikely they would participate in a further 2 h of PA outside of preschool. However, no study has systematically checked to see whether there is a difference in physical activity between weekdays, when the child attends preschool, and weekend days, when the child is influenced more by their home environment. O'Dwyer et al. [21] reported that there were discrete periods during the after-preschool hours and at the weekend when PA levels were low, yet children who attended preschool for full days engaged in 11.1 min MVPA less than those attending for half days, suggesting that the preschool environment is related to decreased PA. That said, studies have shown that PA in preschoolers differs over the course of the day and the week in countries such as Sweden, England and Denmark [21–24], with some studies reporting that preschool children are more often physically active on weekend days than on weekdays in Australia and England, for example [21,25], while others found that preschool children undertook more total PA and MVPA during preschool hours in Sweden, Denmark, England and Finland [22,24,26,27]. Therefore, additional research is required to identify any potential differences in PA between weekdays and weekend days in preschool children. The accurate measurement of PA is fundamental in evaluating the effectiveness of interventions and understanding relationships between PA and health [28]. Measuring habitual PA accurately is beneficial when observing the frequency and distribution of PA in preschool children and identifying the amount of PA that could influence their health. The objective monitoring of PA is important and accelerometers have become a reliable and valid way of estimating children's PA [29,30], whilst also showing promise in monitoring preschool children's PA. Accelerometers are an appropriate objective measure in terms of validity, reliability and practicality as a method for the measurement of intensity, duration and frequency of movement for sedentary behaviour and habitual PA in 3–5 year olds [1,31–33]. Accelerometers can be set at different sampling intervals, with some studies being set at one-minute intervals [1,34]. However, one-minute sampling intervals may mask the short intermittent bursts of activity that are representative of young children and therefore shorter sampling intervals have been recommended [20,35]. As very few studies have used objective monitoring of PA via accelerometery in preschool children, then further research is required to examine the intensity of PA that these children participate in on weekdays in preschool and at the weekend. This is the first study to compare PA levels between week and weekend days, using objective measurements in the form of the newly calibrated GENEActiv accelerometer cut-points for preschool children in the UK. This study aims to determine whether the intensity and duration of PA varies between weekdays and weekend days. #### 2. Materials and Methods #### 2.1. Participants and Data Collection Participants in this study were preschool aged children from 11 preschools in North Warwickshire, England. This study was completed in the Nuneaton and Bedworth Borough, which is in the top 10% of the most deprived Super Output Area's in England on the Index of Multiple Deprivation (IMD) and is ranked as the 111th most deprived Local Authority District out of 326 in England [36]. Ethics approval (P45654) was granted by the Faculty of Health and Life Sciences Ethics Committee, Coventry University and parental consent was obtained. The participants were a convenience sample and included 185 preschool children (99 boys, 86 girls), aged 3–4 years, from a deprived area. #### 2.2. Anthropometric Assessment Height was measured to the nearest mm, in bare or sock feet, using a standard portable stadiometer (Leicester height measure, Leicester, UK). Body mass was measured to the nearest 0.1 kg using portable weighing scales (Tanita scales, Tokyo, Japan); the children were lightly dressed (t-shirt and light trousers/skirt) and barefoot or in socks. The measurements were repeated twice and the average score was recorded. Body mass index (BMI) was calculated as kg/m2 and weight status was categorised as overweight/obese or normal weight using standardised international cut-points [37]. #### 2.3. Assessment of Physical Activity Daily total PA was measured using a GENEActiv waveform triaxial accelerometer (ActivInsights Ltd., Kimbolton, UK). The accelerometer measured at 10 epochs (s) and a sample frequency of 100 Hz, so as to enable an accurate assessment of the intermittent activities of preschool children [38–40]. The GENEA accelerometer was attached using a watch strap and positioned over the dorsal aspect of the right wrist, midway between the radial and ulnar styloid process. Accelerometers worn on the wrist are more convenient to wear and lead to greater compliance during prolonged wearing when assessing habitual activity [41]. The participants wore the accelerometers for four consecutive days; this included two weekdays in the setting and two weekend days. Each child was required to wear the accelerometer for a minimum of 6 h per day to be included in the study, although it was preferred that they wore them at all times. All children received a letter to take home describing how and when they should wear the GENEActiv accelerometers. Non-wear time was defined as 90-minute windows of consecutive zero or nonzero counts [42]. "Nonzero" counts are caused by artefactual accelerometer movements during non-wear periods—for example, accidental movement of the accelerometer, such as the device being nudged when on a bedside table [42]. The 90-minute window was chosen as this was found to better predict time spent in sedentary behaviours and PA levels [42]. This said, Esliger et al. [43] suggested that a period of 20 min of consecutive zero counts is appropriate for children, as motionless bouts of ≥20 min are biologically implausible. However, it was reported that this low threshold causes an unrealistically high number of non-wear periods [44]. Therefore, it was recommended to use 90 minute consecutive zero counts, as this prevents the overestimation of non-wear time and the underestimation of sedentary behaviours in overweight to obese children [42]. The amount of wear time and percentage (%) of wear time that each child spent in different intensities of PA was calculated for weekdays and weekend days. It is recommended that four days, including one weekend day, is appropriate for measuring habitual PA [45]. Given the logistics of ensuring that children aged 3 years of age wore the accelerometer for the whole monitoring period, participants were included in the final data analysis providing they had worn the accelerometer for 3 days (when in the setting and at the weekend) and for a minimum of six hours each day, similar to previous research [46–48]. Of the 185 sample, 178 children's accelerometer data were recorded; data for seven children were not useable. This was due to the children either not wearing the GENEActiv accelerometers or technical difficulties with the accelerometers or recording of the data. The final sample included in the analysis was 178 children (95 boys, 83 girls), aged 3–4 years. Only four out of the 178 participants were full-time; the remainder were part-time nursery attendees. For each of the epochs (number of seconds), movement data (activity counts) were added and logged; these were then processed and analysed. Accumulated activity counts were categorised in terms of intensity such as sedentary behaviour, light, moderate and vigorous PA [1]. Cut-points for sedentary behaviour, light PA and moderate and vigorous PA were used to determine the PA intensity of the preschool children. The cut-points used were determined specifically for children aged 4–5 years using GENEA accelerometers, albeit they were calibrated in a laboratory-based study; they are the most relevant cut-points for the preschool children's age in this study (3–4 years) as they are the closest cut-points that are calibrated and reported in the literature [49]. The difference in age should have very little impact on the results, as they are as closely aligned in age as possible and 4 year olds/preschool children have been used for the calibration and ultimately are assessed in this study. The preschool children in the laboratory-based study that was used to determine cut-points for this current study completed six activities, which ranged from lying supine to running. They wore the GENEA accelerometers on both their left and right wrists and used a Cortex mask for gas analysis; VO2 data were used to assess criterion validity [49]. The cut-points determined were as follows: dominant hand <8.1 cpm for sedentary activity, 8.1–9.3 cpm for light activity and 9.3+ cpm for moderate and vigorous PA. For the non-dominant hand, the cut-points were <5.3 cpm for sedentary activity, 5.3–8.6 cpm for light activity and 8.6+ cpm for moderate and vigorous PA [49]. On the accelerometers, the 'Epoch Converter' creates epochs of 1, 5, 10, 15, 30 or 60 s; the means that for each parameter, the Sum Vector Magnitude is calculated for each epoch [50]. Children were classified as either meeting (sufficiently active) or not meeting (insufficiently active) the requirement of 180 min per day of PA for 0–5 year olds. #### 2.4. Statistical Analysis The percentage of time in sedentary behaviour, light PA and MVPA was determined, as was the mean amount of time (min) spent in sedentary behaviour, light PA and MVPA, during week and weekend days. Each data set was tested for skewness and kurtosis. Arcsine or inverse data transformation techniques were then used on any data set that did not have a normal distribution, as follows: mean time sedentary for the week and weekend (arcsine transformation); MVPA at the weekend (inverse transformation); percentage time for sedentary behaviour at the weekend (inverse transformation); and MVPA at the weekend (inverse transformation). Any differences in PA due to sex or day of the week were analysed using a series (separate ANCOVA for each category of PA) of 2 (weekday vs. weekend) × 2 (sex) repeated measures analysis of covariance (ANCOVA) controlling for wear time. The Statistical Package for Social Sciences (Version 22, SPSS Inc., Chicago, Ill, USA) was used for statistical analysis and the alpha level was set a priori at P = 0.05. #### 3. Results Descriptive characteristics, including mean time (min) spent in the different intensities of PA during the week and weekend days, are summarised in Table 1. Of the sample, none of the 178 children met the UK recommended 180 min or more of PA (light, moderate and vigorous intensity) per day. Two, circa 1%, of the children did meet the 180 mins on one of their days, but not on all days. Preschool children respectively spent 91.9% and 1.8% of time in sedentary behaviour and light PA on weekdays, and 96.9% and 1.1% of time in sedentary behaviour and light PA at the weekend. During weekdays and weekend days, 6.3% and 2.0% of time was spent in moderate and vigorous PA, respectively. The percentage (%) of daily time spent in different intensities of PA during weekday and weekends for preschool children can be viewed in Figure 1. Figure 1. Percentage (%) daily time spent in different intensities of physical activity. (a) Weekdays and (b) weekends for 178 preschool children. There was a significant difference in the percentage of time (relative) spent in sedentary behaviour between week and weekend days (P < 0.05), yet no differences were found between week and weekend days for light or MVPA (Figure 1). There was a significantly smaller mean time in minutes spent in sedentary PA (mean difference = 91.874, P = 0.001) during weekdays compared to weekends. This pattern was reversed for moderate and vigorous PA (mean difference = 4.545, P = 0.001), with a larger mean time spent in vigorous PA during weekdays compared to weekend days. Wear time had no effect on PA, as there was no significant interaction between wear time and weekday (F = 1.308, P = 0.257) or between wear time and weekend day (F = 1.107, P = 0.297) for the significant difference reported for the percentage of time (relative) in sedentary behaviour. Sex had no significant effect on any PA intensity (all P > 0.05). #### 4. Discussion The current study sought to compare PA levels of preschool children between weekdays and weekend days, and the key finding of this study is that there are significant differences in PA between weekdays and weekend days. Of particular note, more than 90% of the time during both weekdays and weekend days was spent in sedentary behaviour. Additionally, this study found that none of the children were considered 'sufficiently active', failing to participate in the UK recommended level of at least 180 min of light PA and MVPA per day of total PA for health. As zero preschool children in this study reported meeting the PA guidelines, this is a major concern, especially as the children were struggling to achieve 60 min of total PA. As the majority of this sample (98%) were part-time preschool children, this could be reflective of this specific preschool child. The children in this study spent time in both the preschool setting and with their parents, in the home environment. It would be pertinent to assess preschool children who are full-time, to ascertain if this provides a similar or different outcome in their PA levels. This would be influential in identifying whether the results of this study are reflective of British preschool children, or if the time children spent in preschool affects their PA levels. Studies have shown that parents have a significant impact on PA in their preschool children on week and weekend days [51–54]. Therefore, other explanations for the differences in sedentary behaviour between the week and weekend days could be a result of parents displaying higher sedentary behaviours when they are with their children, and children who are exposed to these behaviours copy them during weekend days [55]. This is supported by Sigmundová et al. [56], who found that children from both urban and rural populations had a stronger significant association with sedentary behaviour during weekend days as compared to weekdays. Moore et al. [57] discovered that middle-class American children aged 4–7 years old (incorporating the preschool years), who have one physically active parent, had a relative odds ratio of their child being active between 2.0 (mother) and 3.5 (father). However, if both parents were active then the relative odds ratio was 5.8, with no difference reported between week versus weekend day. Sigmundová et al. [56] monitored children from both urban and rural areas, and discovered that if mothers are more active, then their children are more likely to be more physically active. This was observed to be significant only on weekend days. We do not know the activity patterns of the parents of the children who took part in the present study, but this may have been a contributing factor to the sedentary behaviour patterns that were reported. A second key finding of this present study is that there are differences in the percentage of time spent in different intensities of PA between weekdays and weekend days for preschool children. During weekdays, the children spent significantly less time in sedentary behaviours (91.9% vs. 96.9%), when compared to weekends. This finding was recorded using the right wrist, which in this study was the dominant hand for 169 of the children (91.4% of the sample). This finding contradicts research by Vásquez et al. [58] in Chilean children (North of Santiago City), who objectively measured PA via a Tritrac-R3D research ergometer and reported that preschool children spent more time in sedentary activities in day-care centres (week vs. weekend) and the children were more active at home at weekends. These differences were also linked with the children's diet and it was discovered that the energy balance was appropriate during the week, as the energy intake in the preschools was reduced. This could explain the differences, as the day-care settings were providing less energy intake; therefore, the children may have been less inclined to be active. Equally, these findings could have been representative of the cultural background, geographical location or differences in the method used for objective measurement of the children involved. A further reason for potential differences between the sedentary behaviours of preschool children during the week and weekend days could be attributed to the time that children spend watching television or playing on computer games, smart phones/tablets (screen time). Previous research has shown that Australian preschool children from different socio-economic backgrounds whose parents limit television viewing spent significantly less time in sedentary behaviours [59]. A study of preschool children from a mixed socio-economic area in the southwest of England reported that 12% of boys and 8% of girls watched ≥2 hours of TV each weekday, compared to 45% of boys and 43% of girls who watched ≥2 h a day on weekend days [60]. The amount of screen time that children in this current study participated in could have been a contributing factor to the differences in time spent in sedentary behaviours during week and weekend days. Parental influence on PA during weekend days may therefore be an important factor that requires greater attention by public health professionals; this could be a result of parents lacking an understanding of appropriate PA to deliver to their children, or a lack of time. Despite this, the extant literature on parental influence on PA levels in British preschool children is scarce. Additional work is required on this topic, in the context of weekday to weekend day variations in children's PA. Equally, the intensity levels of PA of preschool children could be improved through interventions, both in preschools and at home with parents. Research has reported PA levels and sedentary time as being highly varied and inconsistent between studies across different countries, making it hard to determine preschool children's true PA levels and sedentary behaviour [61]. Reilly [12] measured PA levels from studies over the period of 2000–2008 and discovered that sedentary behaviour was particularly high. However, more recently, in a data collection period from 2006–2009 and a 7 month data collection period in the year 2013, it was reported that 100% of UK preschool children met the recommend daily PA guidelines [22,23]. The current data from this study show that a substantial proportion of each day is spent in sedentary behaviour in British preschool children from a deprived area. This finding has important public health implications around the excessively high sedentary behaviours displayed by preschool children in the UK and therefore provides a clear indication that interventions aiming to convert sedentary behaviours into light or MVPA are required. These data were obtained across a wide measurement period and across all seasons and therefore provide a spread of representative data for the whole of the year. However, this study did not assess each child at different points throughout the year, as it is very labour-intensive and demanding to assess PA in preschool children at one time point and then assess them again across different seasons, and would likely lead to much higher attrition. Therefore, this study did not consider seasonal adjustments. However, future research may consider this, to identify if preschool children are more active in the summer months in England, when compared to the cold environment in the winter months, similar to the results found in 437 preschool children in America [62]. However, this was in contrast to a study of 214 children aged 3–5 years in America, which reported no differences in PA levels between the summer and autumn months [63]. Moreover, further studies in Sweden and America [64,65] also found no variation according to the season of measurement in preschool children. As none of these studies were conducted in England, future research into the seasonal variation of PA in England would be beneficial. The amount of time spent in different intensities of PA was found to vary between weekdays and weekends, with less moderate and vigorous PA accrued during weekends. However, MVPA was very limited in both parts of the week. Such a finding might be suggestive that regular engagement in the preschool environment provides greater opportunities to accrue PA, which may not be present in the home setting. The light intensity minutes were very low during both the week and weekend days. The wrist-worn accelerometers may not be very precise at detailing light ambulation, where playing with Lego®was at the top end of the sedentary category. Therefore, future research to determine the accuracy of the light PA classifications would be beneficial. Similar research using Actigraphs has shown that using the cut-point of 160 cpm to distinguish light intensity from sedentary behaviours is questionable [66]. It is believed that this threshold may misclassify sedentary behaviours such as seated play and crafts as light intensity, which would cause an overestimation of total PA minutes and underestimate the steps per day [66] that are required to achieve the daily UK 180 minutes of recommended PA. This is a current problem, as there is no consensus on the optimal cut-points for distinguishing sedentary from light PA in preschool children [56]. Also, the cut-points used for light intensity PA in this study were taken from laboratory-derived tasks that were constant in nature, whereas the real-life activities of preschool children are more varied and intermittent. This may have made it more difficult to differentiate light PA from sedentary behaviour, as the determination is dependent on the cut-point used. However, this is a feature of most of the research using accelerometers to classify/assess PA [66,67]. Therefore, it could be suggested that the cut-points used in this study were too conservative, however, as they are the only cut-points related to preschool children specifically for the GENEActiv accelerometers, then they were the most appropriate to have adopted. Using wrist-worn accelerometers can be logistically and practically challenging with preschool children [31], as the accelerometer can sometimes be regarded as uncomfortable or an annoyance when worn for long periods of time, thus questioning the appropriateness of the accelerometer for preschool children. In the current study, although a cut-off of 6 h per day was employed for inclusion in the data analysis, the participants exceeded this value with total mean wear time per day for all days, which was over 577 min (>9.6 h). The mean wear time for weekdays was 573 min (9.6 h) and that for weekend days was 581 min (>9.7 h). We took this to be an indication that the majority of the children in this study were comfortable wearing the accelerometers. It could be questioned whether <600 min/day (10 h) is a true representation of a preschool child's whole day (24 h). It would be beneficial for future research to measure PA for a greater duration, for example 12 hours, to see if this affects the total PA in a day, in terms of less sedentary behaviours and more MVPA. As mentioned, it could be questioned whether wearing the accelerometer on the wrist is suitable for preschool children. Research has looked at the difference between wearing a wrist and hip accelerometer on preschool children and one study in Scotland found that wrist-worn accelerometers provided a valid estimate of total physical activity, whereas hip-worn accelerometers showed a reasonable agreement to cut-points [68]. This was supported by a study in Stockholm, Sweden, which similarly monitored preschool children and found that wrist-worn accelerometers performed more accurately when assessing time spent in sedentary, light activity and MVPA, when compared to hip-worn accelerometers [69]. In terms a study by Johannsson et al. [69], however, there were stark differences between the mean (SD) counts measured over 5 s for wrist and hip activity, with the vector magnitude (a combined measure of the three axes, x, y and z) when watching a cartoon measuring 91 (73) for the wrist and 14 (15) for the hip, and when dancing, the 1093 (330) was measured for the wrist and 396 (148) for the hip. These are large contrasts, highlighting that where the accelerometers are worn is an important factor to be considered. This present study had the preschool children wearing them on their wrist, which, in accordance with other studies [68,69], is the more accurate and valid place to wear them when using the vector magnitude measure, as opposed to the vertical axis measure. Although the current study successfully used accelerometery as an objective monitoring tool in preschool children, some limitations should also be considered. Some of the accelerometers failed to record data; this manufacturer problem caused no data to be recorded for seven participants. GENEActiv accelerometers worn at the wrist may not be capable of detailing light ambulation precisely, as possibly indicated by the low levels of light PA reported. Equally, wrist-worn accelerometers in young children may also impact the light intensity PA data; however, the research does appear to show that the location, whether at the wrist or hip, has no significant effect on the PA levels reported [70,71]. As previously stated, the preschool children were assessed once in a season and not across all seasons. Although children were assessed throughout different seasons, which provided representative data, the lack of assessment of each child in all seasons should be considered a limitation of the study. The preschool children that were monitored were drawn from a deprived part of the UK. It has been reported that the prevalence of obesity amongst 4–5 year olds in the most deprived 10% of England is approximately double the levels of the least deprived 10% of England [72]. In this current study, 10.8% (20 out of 185) were considered obese. Low socio-economic status (SES) children face greater barriers to becoming physically active and, as they age, low SES individuals have higher rates of obesity and associated comorbidities [73]. Additionally, people from lower SES groups predominantly live in areas that do not support walking and cycling [74]. This viewpoint suggests that deprived areas do not facilitate PA as effectively as other areas and, as such, people living in deprived areas may not participate in PA as frequently. This said, further research comparing both high and low socio-economic status groups would be welcome to extend the literature on preschool children. Understanding the levels of PA in this group is useful to allow for the planning of early interventions to improve current and future health. #### 5. Conclusions The current study is the first to objectively compare PA levels between weekdays and weekend days in preschool children in the UK using GENEActiv accelerometers, and one of the first to report objectively monitored PA levels of preschool children from deprived areas in the UK. The results of this study suggest that none of the preschool children in this sample achieved the UK recommended guidelines of PA for health. Additionally, and of great concern, it was found that preschool children spend 90% of their time engaged in sedentary behaviours. This study indicates that preschool children participate in more MVPA during weekdays compared to weekend days; however, participation in MVPA was minimal throughout the week. This information can help to promote future interventions that focus on enhancing PA and encouraging participation in LPA and MVPA during both week and weekend days, so as to improve physical development and a healthy weight status in preschool children. Author Contributions: C.M.P.R.: conception and design of the study, data acquisition (participant measurements), analysis of the data, preparation of the tables and figures, preparation of the manuscript, finding relevant references and final approval of the manuscript. M.J.D.: conception and design of the study, analysis of the data, writing—review and editing and final approval of the manuscript. R.S.J.: analysis of the data, writing—review and editing. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). #### Article	doab	2025-04-07T03:56:58.863985	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 14 }
0051d55d-461b-414c-a51b-301e45ff4d0c.15	Joint Mobility Protection during the Developmental Age among Free Climbing Practitioners: A Pilot Study Ludovica Gasbarro 1,†, Elvira Padua 1,† *, Virginia Tancredi 2, Giuseppe Annino 2, Michela Montorsi 1,\ , Grazia Maugeri 3,\* and Agata Grazia D'Amico <sup>1</sup> Received: 5 September 2019; Accepted: 10 February 2020; Published: 17 February 2020 Abstract: Sport-climbing popularity increased intensely over the past years. Particularly, children's and adolescents' interest therein is constantly growing. Despite a large effort in preventing injuries and muscle overloads, a fine-tuned training for each sensitive phase of child development is still needed. The objective of the study was to evaluate an innovative training program aimed at the preservation of joint mobility during the developmental age. This article relies on the results of a steady training program allowing to retain joints integrity among the practice of sport climbing in children. Joint mobility changes have been monitored before and after a one-year training program in fifteen subjects aged between 8 and 18 years. Subjects were divided into three groups depending on age (Turgor Secundus, Proceritas Secunda and Turgor Tertius). The motor tests administered were the sit-and-reach test, coxo-femoral mobility test and scapula–humeral mobility test. Our results showed that one-year training improved joint mobility at each analyzed phase, suggesting that this training program could improve mobility and flexibility. Given the importance of joint mobility preservation for discipline-related injuries prevention and eventually recovering, it is essential to provide a specific training program as a route to approach sport climbing, and even more importantly, at an early age. This work represents a preliminary study in order to demonstrate both efficacy on the joint mobility and the requirement of our playful work to support the global sport-climbing workout. Keywords: joint mobility; development phases; sport climbing; stretching #### 1. Introduction In the last decades, rock climbing has become a popular sport among adults, adolescents and children, also since it being included in the 2018 Summer Olympic Youth Games in Buenos Aires and in the Summer Olympic program of the 2020 Games in Tokyo [1,2]. In using only their bare hands and climbing shoes to perform a range of hand and foot holds, athletes climb vertical walls in three disciplines: Speed, with two climbers simultaneously climbing a route on a 15 m wall; Bouldering, with athletes performing in a given time a number of fixed routes on a 4m wall; and Lead, with athletes climbing in a given time a 15 m wall [3]. It is possible to practice it not only outdoor, as now there also are indoor structures; so, this sport is becoming widespread in adults and adolescents, too. Rock climbing has several benefits on both physical fitness and mental care. In fact, this sport improves strength and endurance [4,5]. A recent study [6] has demonstrated that rock climbing significantly improves muscle power, ability to produce a maximal force in a short time, muscle endurance and skills to perform a continuous muscle work for a long time. This kind of sport also has beneficial effects on depressive disorders due to the positive effect on cognitive control of the physical activity connected to high levels of coordination [7]. It has been demonstrated that only a single rock-climbing session may have a positive effect in major depressive disorder [8]. Due to the increased popularity, the average age of rock climbers is decreasing, and more and more young athletes win medals. Unfortunately, the increased number of young rock climbers involves a major risk of an injury in the developmental age [9]. During climbing very small parts of the hands and feet are in contact with the climbing surface [10–12] and climbers have to support and/or lift their bodies by combining a variety of finger grips with balanced, complex vertical and lateral movements and position holds [13,14]. To guarantee grip and stability, specialized smooth climbing footwear with a sticky rubber sole are used. Although climbing shoes should fit snug without pain, the climbers tend to use smaller sizes, which could cause injuries or foot deformation [15]. Physical activity is essential to maintain good health and guarantee a better quality of life. The major sport-related benefits involve not only the body but also the mind. In fact, physical activity improves motor and cognitive skills, reduces risk for obesity, exerts positive effects on blood pressure and lipidemia, as well as decreasing the risk of depression and other mental disorders [16,17]. However, incorrect or excessive training may lead to adverse effects, including musculoskeletal injuries, recently described in young soccer players, as well as social–occupational dysfunction [18,19]. In particular, in rock climbing the articulations are very solicited and the scientific literature reports injuries in fingers or in shoulders [15,20]. In particular, Garcia et al. [21] examined hands and fingers of young climbers versus a control group of non-climbers and they showed a non-physiological development of fingers. Similarly, another study [22] showed differences in the static scapular position between rock climbers and a control group. In particularly the developmental age there is a major risk of sprains, strains and fractures with chronicle injuries at the upper extremity and acute injuries at the lower extremity [23]. A survey among rock climbers [24] reports 90% upper extremity injuries, and among these the most common are fingers followed by shoulder/arm and elbow/forearm. Rock climbing is positively related to increased bone mineral content, weight and mass body [15]. However, for climbers a low body mass index is an ideal anthropometric requirement. Therefore, they perform a restricted diet to maintain a low body weight, by inducing a negative effect on their health and in particular on their bones [25,26]. Moreover, the stresses associated with rock climbing may have the potential to create scapula–humeral or coxo–femoral injuries [22]. Despite the increase in rock-climbing practices and the consequent increase in studies on the benefits or injuries of climbing, little data are available on adolescents. To envision the development of effective preventative measures for preserving the joint mobility and health of youth practitioners, the aim of this preliminary study was to evaluate the effects of a specific pre-training rock climbing program to be administered to athletes according to their developmental status. Therefore, in this preliminary study we evaluated an innovative training program to preserve joint mobility during the developmental age. Children were subjected to a steady training program in order to retain joint integrity and to avoid climbing-associated injuries. #### 2. Materials and Methods** #### 2.1. Subjects Fifteen subjects aged between 8 and 18 years, working out regularly in sport climbing and joint mobility, were divided into 3 groups consisting of 5 athletes each according to their ages. Turgor Secundus (3 boys and 2 girls; aged between 8 and 10), Proceritas Secunda (2 boys and 3 girls; aged between 12 and 14) and Turgor Tertius (2 boys and 3 girls; aged between 15 and 18). Table 1 reports the anthropometric characteristics of the athletes, divided in subgroups according to their developmental phase. Table 1. Characteristics of participants recruited to conduct the present study expressed as mean fold change ± SEM. This study received the consence by the Institutional Research Board of the University San Raffaele of Rome, Italy and all subjects' legal tutors gave written informed consent in respecting the ethical principles of the Declaration of Helsinki. All participants were novices regarding climbing experience. #### 2.2. Methodology Knowledge of the main physiological changes and the sensitive phases of development is fundamental for the educator, as on the basis of this information he can develop a program suitable for the child's needs. The sensitive phases of development are periods of growth in which the child will be predisposed to increase some motor skills rather than others. Furthermore, the differential growing of the bones, nervous system and muscles in the different evolutionary phases must be taken into consideration in the programming to avoid injuries and the onset of paramorphism. Our study was conducted for 12 months, and during this experimental period the climbing training program was carried out according to the coach's training plan. Children worked out twice a week for 90 min divided as follow: 15 min for stretching, 20 min for specific training to improve joint mobility and 55 min for climbing. Joint mobility variation was assessed over a one-year training period. All the measurements were performed at baseline (time 0; start measurement) and repeated at the end of the training period (time 1; after 12 months) in order to evaluate scapula–humeral and coxo–femoral joint mobility and spine flexibility. The measures recorded in time 0 were considered our control data and the measures obtained at the end of training program were compared to data recorded in time 0 in order to calculate the improvement of joint mobility for each group. Tests have been performed as follows: Sit-and-reach test: Participants sat on the floor with legs extended, backs straight and feet resting on a cube with a graduated wooden board above it. The participants are asked to slide their hands above the wooden board, keeping the knee extended [27]. Hip joint mobility: Participants are sitting with their back against the wall, slowly reaching the maximum opening of their hips. The measurement is then taken between the inner ankles [28]. Scapulo–humeral mobility test (measurement of shoulder mobility): Participants are in an upright position, holding a stick, bringing the arms outstretched behind the trunk and reaching the starting position without bending the arms. Then, the minimum distance between the hands while the subject is holding the stick was measured [29]. #### 2.3. Training Regime Participants of the three experimental groups underwent a specific training program 2 times/week, performing 15 min of general warm-up before training. The specific training program for each phase was done for 20 min before rock climbing. According to each phase, the following training methods were proposed in order to improve joint mobility. The Turgor Secundus phase: Obstacle course on wall bars (Figure 1) to preserve a good degree of flexibility and to practice the typical positions of sport climbing: Exercise 1: The aim was to start from the left side of the wall bar and moving sideways to pass over, under and through some obstacles, following a predetermined path, without falling (Figure 1). Figure 1. Children performing exercises starting from the left side of the wall bar and moving sideways to pass over. Exercise 2: The game of spiders and crabs: half of the children have to positioning themselves in a line, keeping their arms and legs outstretched, forming a large arch. The crabs will have to come through it (Figure 2). Figure 2. Overview of spider and crab positions. The Proceritas Secunda phase: Mobilization of the spine (Figures 3 and 4a,b) and the tibio–tarsic joint (Figure 5): Exercise 1: Hamstring stretch involving two subjects (A and B) that have to sit back-to-back on the floor with their legs extended forward. Subject A is stretching back by abducting his/her arms upwards slightly pressing its weight, and simultaneously subject B reaches for his/her toes. The position has to be kept constant for 4–5 s, after which this exercise is repeated but with the subjects changing their roles (Figure 3). Figure 3. Final position of the spine mobilization exercise. Exercise 2: The subjects sit up tall in the straddle position feet-to-feet, with straightened legs, and holding a ball in their hands (Figure 4a). They slowly lean down on the back, closing their legs and curling the pelvis inward until their toes touch the ball behind their heads, and then back to the starting position (Figure 4b). Figure 4. (a) Starting position of the mobilization of the spine and coxo–femoral joint exercises. (b) Ending position of the mobilization of the spine and coxo-femoral joint exercises. Exercise 3: In single foot support, rotate the rope forward without jumping. After rotating the rope forward, to pass the rope beyond the foot, flex the foot and then extend it. Do the same with the other foot (Figure 5). Figure 5. Flex foot and rope locked by the foot. Turgor Tertius phase: Mobility exercises for the fingers (Figure 6), coxo–femoral (Figure 7) and tibio–tarsic joints (Figure 8): Exercise 1: Starting with a hand fully opened; perform four closing finger movements, through which both the metacarpophalangeal and the interphalangeal joints will be stimulated (Figure 6). Figure 6. Mobility exercises for fingers from starting to ending positions. Exercise 2: The subject is crouched down, the feet are in a wide stance with the toes turned out, the back outstretched, the heels on the ground and the elbows slightly pushing the knees outwards. Then, small rotations of the ankles are made first in one direction and then in the other (Figure 7). Figure 7. Squat position for mobilization of the coxo–femoral joint. Exercise 3: Four different types of gaits are proposed. First, toe walking with raised arms. Second, heel walking with raised arms. Third, foot rolling forward. Lastly, feet supination and pronation walking forward (Figure 8A–D). Figure 8. (A) Toe walking with raised arms; (B) foot rolling forward; (C) heel walking with raised arms; (D) feet supination and pronation walking forward. #### 2.4. Statistical Analysis Data collected at baseline (pre-training) and after (post-training) the one-year experimental period are presented as the mean ± SD. The assumption of normality was verified by means of the Kolmogorov–Smirnov test. Then, for each subgroup a paired t-student test was used to ascertain differences between pre-training and post-training data. Throughout the study, the level of significance was set at p ≤ 0.05. Statistical analysis was conducted using GraphPad Prism version 6. #### 3. Results Athletes have carried out the proposed activities for the entire duration of the study over and above the regular workout, with the exception of a female belonging to the group Proceritas Secunda who interrupted the training program for one and a half months. However, data from subjects who dropped out were used for preliminary comparisons. All groups considered in our study have shown an improvement in joint mobility (Table 2). However, differences in training response among the considered phases have been identified. Table 2. Percentage of improvement in flexibility in the different phases expressed as mean fold change ± SD. Proceritas Secunda showed a positive response in coxo–femoral mobility and lower vertebral column flexibility 12 months after the start of the training program (Figures 9 and 10), whereas scapula–humeral mobility did not report benefits after joint-specific training (Figure 11). On the other hand, scapula–humeral mobility remarkably improves in the Turgor Tertius phase when compared to both Turgor Secundus and Proceritas Secunda (Figure 11). Indeed, this is consistent with the normal development of the shoulder joint that tends to complement the most advanced evolutionary phases. However, this does not apply to the other two phases (Figure 11). The Turgor Tertius group have also recorded an improvement in spine flexibility as reported in Figure 10. Regarding Proceritas Secunda, we further compared two 14-year-old subjects (Figure 12). Athlete A has completed his/her training, whereas Athlete B has totally interrupted the workout for one and a half months. The latter has been previously excluded from the overall analysis. It is noteworthy that we clearly identified a remarkable impairment in terms of mobility and flexibility as a result of the training discontinuation of Athlete B. Figure 9. Coxo–femoral mobility test. The results are represented in bar graphs as the mean ± SD of the different phases considered (Turgor Secundus, Proceritas Secunda and Turgor Tertius). Figure 11. Scapulo–humeral mobility test. The results are represented in bar graphs as the mean ± SD of the different phases considered (Turgor Secundus, Proceritas Secunda and Turgor Tertius). Figure 12. Comparison between the percentage mean variations of two athletes: Athlete A has completed the training, while Athlete B has totally interrupted the workout for one and a half months. #### 4. Discussion This work represents a preliminary study in order to demonstrate both the efficacy on joint mobility and the requirement of our playful work, to support the global sport-climbing workout. Indeed, the increased interest in rock climbing practices creates the need to know all the possible sport-related injuries in order to prevent and treat them. Hence, the importance to study the main joints involved in climbing and that are at risk of injuries, such as joints of the hand and fingers, shoulders, the hip and ankles, in order to develop a preparation workout suitable for the discipline. Since sport climbing does not need specific anthropometrical characteristic, and for this reason it can be practiced by everyone [30], a specific training program in this sport can improve one's strength and resistance; essential skills that would allow to carry out increasingly difficult passages. In fact, climbers perform movements that require an isometric effort, resulting in an enhanced muscle tone to obtain a strong and harmonious body. Moreover, although it is not an aerobic discipline, it develops a good cardiovascular training; this is also due to the adrenaline that develops while climbing [31]. Because of a child's incomplete maturation of both bone and muscular structures, and the laxity of ligaments, early and specific training would reasonably provide extensive room for improvement. We have proposed a playful training workout as a mobility workout in the first sensitive stages. In the present work, we have conducted a preliminary study in an attempt to prove that a specific training program is fundamental for mobility and flexibility improvement. Participants enrolled for our study were novices regarding climbing experience and they worked out twice a week for 90 min in agreement with the coach's training plan for 12 months. Although the reported data did not reach statistical significance, we have recorded an improvement in joint mobility after the playful training program. However, not all the recorded ameliorations were statistically significant. This might be due to external factors affecting joint mobility, such as anatomical and physiological differences among subjects. However, this can be also ascribable to the low sample size. The latter stems from the direct correlation between the standard deviation value and the sample size. Moreover, by comparing continuous and discontinuous training between two athletes, we have suggested the importance of early and constant training (Figure 12). Indeed, the suspension of a joint-specific workout can lead to a blockage or even a regression of the subject's abilities. Thus, injuries require prompt treatment in order to avoid transient and permanent physical impairment. Even if the study was conducted on a low number of subjects, this represent a starting project in order to develop a specific training program for each developmental phase. #### 5. Conclusions The recent proliferation of indoor climbing gyms and well-protected sport climbing areas have made sport climbing accessible to everyone. Neither an age group nor a pre-existing medical condition serves as a contraindication for sport climbing in the first instance. Sport climbing is an engaging mental and physical activity that contributes to an increase in muscle mass and strength, dynamic balance, and other health benefits. However, in line with any exercise prescription, guidelines for sport-specific participation are desirable. Taking into account what was shown in this preliminary study, early and constant joint-mobility training that start from the early stages of the developmental age is fundamental. In conclusion, we suggest that joint-mobility exercises from the early stages of development will allow children to move harmoniously, even on the climbing walls. Author Contributions: Conceptualization, L.G. and A.G.D.; methodology, L.G.; validation, E.P. and G.A.; formal analysis, M.M. and V.T.; investigation, L.G.; data curation, A.G.D.; statistical analysis, G.M. and A.G.D.; writing—original draft preparation, M.M., A.G.D. and E.P.; writing—review and editing, M.M.; G.M. and V.T.; supervision, A.G.D. All authors read and approved the final manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). ## Article Cognitive and Physical Activity-Related Aspects of Children Associated to the Performance of the Crunning Movement *Ewan Thomas 1,\ , Marianna Alesi <sup>1</sup> , Garden Tabacchi <sup>1</sup> , Carlos Marques da Silva <sup>2</sup> , David J. Sturm <sup>3</sup> , Fatma Ne¸se ¸Sahin 4, Özkan Güler 4, Manuel Gómez-López <sup>5</sup> , Simona Pajaujiene <sup>6</sup> , Michele Basile 7, Ante Rada 8, Antonio Palma <sup>1</sup> and Antonino Bianco <sup>1</sup> Abstract:** The aim of this investigation was to identify possible related factors associated to the performance of the crunning test in European children and adolescents. A total number of 559 children and adolescents (age range 6–14 years) of which 308 boys (55.1%) and 251 girls (44.9%), from seven European countries, were screened. A questionnaire concerning demographic and personal life-related factors and a cognitive assessment were performed. A regression analysis was conducted with the performance measures of the crunning movement. T-tests and ANCOVA were used to analyze sub-group differences. Boys have greater crunning performance values compared to girls (5.55 s vs. 7.06 s, p < 0.001) and older children perform better than younger ones (R2 −0.23; p < 0.001). Children with healthy and active habits (exercising or spending time with family members vs. reading or surfing the internet) performed better in the test. Children engaged in team sports had better crunning performances compared to those engaged in individual sports (6.01 s vs. 6.66 s, p = 0.0166). No significant association was found regarding cognitive-related aspects in either children engaged in team or individual sports and the crunning performance. Older and male children performed better in the crunning test than younger and female children. Physical activity-related aspects of children's life are associated with crunning movement performance. No association was found between higher cognitive performance and the crunning test results. Keywords: crunning; socio-demographic; cognitive; fitness-tests #### 1. Introduction Physical fitness (PF) during childhood and adolescence has been deeply investigated [1–3]. It has been recognized that PF is an important health-related factor, which may predict health status in adulthood, and which may also help physical and cognitive development [1,4–6]. Inverse associations have been found between PF and cardiovascular disease, metabolic risk factors and adiposity, which overall suggest positive health-related Citation: Thomas, E.; Alesi, M.; Tabacchi, G.; Silva, C.M.d.; Sturm, D.J.; ¸Sahin, F.N.; Güler, Ö.; Gómez-López, M.; Pajaujiene, S.; Basile, M.; et al. Cognitive and Physical Activity-Related Aspects of Children Associated to the Performance of the Crunning Movement. J. Funct. Morphol. Kinesiol. 2021, 6, 9. https://doi.org/10.3390/ jfmk6010009 Received: 16 December 2020 Accepted: 13 January 2021 Published: 17 January 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). aspects [5,7,8]. A recent review by Donnelly et al. [4], investigating the effects of PF on cognition, learning and brain structure and function in children aged 6–13 years old, found a general positive association regarding cognition and brain structure and function. However, the study failed to identify a specific set of activity parameters which may better promote the abovementioned aspects. However, despite different evidence existing regarding a positive association between physical activity (PA) and academic achievements [9], there are still inconsistencies regarding aspects of PA and cognitive function [4]. In order to promote the level of PF through PA in adulthood, it is important to adequately modify lifestyle and behaviours in childhood and adolescence [10,11]. Different investigations have suggested that children and adolescents who did not regularly practice PA are more prone to develop unhealthy habits as smoking or drinking alcohol [12,13], which would inevitably decrease PF. In addition, such aspects have also been linked to the familial socio-economic status, parental education and food habits [12,13]. Therefore, since lifestyle and familial education are important factors which influence both physical and psychological development of children and adolescence, these may be considered as important predictors of PF and health outcomes [14]. In order to quantify the levels of PF, a large body of scientific evidence exists which takes into account specific age ranges and genders [1,15–18], demographic characteristics [19–21] or more specific sub-populations [22,23]. Each investigation has evaluated different aspects related to PF, such as aerobic activity, strength, power, speed, coordination, flexibility, agility and other abilities, including for each of these, one or more field or laboratory tests to appropriately discriminate such abilities. Despite the large number of fitness tests and physical evaluation batteries available, performance values or predictors of the crunning movement, a specific type of locomotion which combines running and crawling, are scarce. Patrick et al. [24] evaluated the inter-limb coordination during crawling in infants and adults, suggesting that a great involvement of coordination by the central nervous systems is required for quadrupedal locomotion. Other two investigations have included specific crawling exercises, within a fitness battery, to improve PF in a military environment [25,26]. The results of the studies underline that the military personnel increased the efficacy of different abilities, such as coordination, agility, speed, and power, following such interventions. Another investigation tried to determine a link between the Illinois agility test, which evaluates agility, and the crunning test. The results indicate a moderate correlation (r = 0.45) between the two tests [27], suggesting that a certain amount of agility is required to perform the crunning movement. In an attempt to understand if the crunning locomotion could provide further information than those offered from already known fitness tests, we included the crunning test in a previously discussed project [15]. This project, namely, the Enriched Sport Activity (ESA) program, was a physical activity intervention with the aim to improve fitness of children and adolescents across Europe by including cognitively enriched stimuli within specific warm-ups, prior to a structured physical activity [15]. However, such tests, together with the Leger shuttle run for aerobic assessment [28], were the only tests not influenced by the ESA intervention. In order to clarify the characteristics of the crunning movement, this investigation will aim to identify lifestyle, physical activity and cognitive aspects associated with the results of this particular type of locomotion. #### 2. Materials and Methods #### 2.1. Participants The sample was composed of 589 children of ages ranging from 6 to 14 years (aged 10.25 ± 1.76 years and 9.98 ± 1.87 years) of which 308 boys (55.1%, age 9.1 ± 1.3 years; weight, 34.9 ± 9.5 kg; height, 139.0 ± 10.4 cm) and 251 girls (44.9%, 10.2 ± 1.8 years; 39.4 ± 11.1 kg; 144.8 ± 14.3 cm), from 7 European countries (Italy, n = 164 of which 92 boys and 72 girls; Germany, n = 64 of which 41 boys and 23 girls; Portugal, n = 111 boys; Spain, n = 37 of which 17 boys and 20 girls; Lithuania, n = 85 of which 53 boys and 32 girls; Croatia, n = 50 of which 25 boys and 25 girls; and Turkey, n = 78 girls) within the ESA Program, an evidence-based exercise program cofounded by the Erasmus + Program of the European Union (Key action: Sport-579661-EPP-1-2016-2-IT-SPO-SCP). Criteria for including participants were the following: 1. Able to perform the required tests; 2. Absence of a diagnosis of intellectual disability, visual or neurological impairments; 3. Absence of a diagnosis of other neurodevelopmental disorders. All children were recruited within a school or sport center. Before the inclusion of the children in the ESA program, a parent or legal representative of each child signed a declaration of informed consent. The study was conducted in accordance with the Helsinki Declaration (Hong Kong revision, September 1989) and the European Union recommendations for Good Clinical Practice (document 111/3976/88, July 1990). The study was approved with permission from the Lithuanian Sports University's Research Ethics Committee in Social Sciences with approval No 579661-EPP-1-2016-2-IT-SPO-SCP (2018-02-05). #### 2.2. Study Design The ESA Program aimed to improve children's motor skills and executive functions through sport activities enriched by cognitive stimuli to enhance inhibitory control, working memory and shifting. Detailed description of the study design and the ESA Program can be found elsewhere, describing both the cognitive tests and their administration and motor components, as well as the full description of the crunning test and administration procedures within the project [15,29]. #### 2.3. Intervention Procedure and Assessment Tools Each ESA trainer underwent a training procedure before the start of the intervention, in order to adopt a standardized procedure. The procedure was shared across the participating countries. This can be also found on a dedicated YouTube channel (ESA Program). Therefore, all testing procedures were performed in the same way and order. At the beginning of the project, each participant underwent a cognitive examination [29] and a physical evaluation [15], which were repeated at the end of the intervention. Before the start of the ESA training program, each participant also underwent a psychological test battery (ESA PTB, a 32 item questionnaire which includes questions regarding personal information, lifestyle-related factors and school-associated information), which was administered on two different occasions prior to the inclusion of the participants in the ESA program. On the first day, the participants underwent a cognitive assessment which lasted around 1 h [29]. On the second day, a questionnaire was administered, which included questions related to habits, family, recreational and social aspects. We retrospectively evaluated the predicting factors of the crunning performance through these personal aspects. The crunning performance, measured as the time in seconds needed to perform the test (10 m distance), was the dependent variable collected through the crunning test, included in the ESA fitness battery. This battery is composed of six fitness tests (standing broad jump, seated medicine ball throw, 20 m shuttle run test, 30 m sprint, Illinois test and the crunning test) to assess different skill-related components at once [30], but for the purpose of the present paper, only the outcomes of the crunning test have been considered [15]. Low values in the test measure indicate better performance. For the purpose of this study, gender and age were treated as covariates, while a number of predictors were taken into account. These predictors included sport-related aspects, spare time-related aspects and cognitive or neuropsychological aspects. Each aspect is discussed below. (a) Sport-related aspects: Sport type (individual/team and exercise intensity) and sport frequency. Individual/team sport type classification was manually distinguished by the authors on the basis of the sport practiced. The American Heart Association classification for sport was adopted [31] for grouping the activities in relation to cardiovascular function. These are divided by static and dynamic activities, and each is stratified according to exercise intensity, categorized as low, moderate or high. Current sport frequency and past sport frequency were measured in hours per week and relevant data were collected through a dedicated section of the ESA PTB. (b) Spare time-related aspects (frequencies in h/week): Aspects regarding time spent with parents and siblings, reading, going to the cinema, theatre or museums, surfing the internet, playing with electronic games, going out with friends, going to the gym, going to the park or shopping were evaluated. All these items were collected through a dedicated section of the ESA PTB. (c) Cognitive/neuropsychological variables: School marks (National language, Maths, Anthropology and History, Geography, Physical education, Foreign language), inhibitory control, working memory, shifting of attention. The ESA PTB was used to collect the school mark items. The other items' assessment was performed through the neuropsychological tests derived by the Inquisit Lab platform (Inquisit 6 (for Windows version 6.1). 2020. Retrieved from https://www.millisecond.com): the Color Word Stroop (CWS) task for the inhibitory control, measured by the time needed to select the right color within the stroop test; the Digit Span Test (DST) for working memory, measured by the number of recalled digits in an exact order and in a reversed order; and the Trail Making Test (TMT) for shifting of attention, measured by error number and execution time. For further details regarding the cognitive test administration, please refer to Gentile et al. [29]. #### 2.4. Statistical Analysis Data are presented as means and standard deviations. The Student's t-test was used to assess differences in the crunning test for each variable (i.e., gender, sport type). To estimate differences in the crunning performance by country, age range and sport type, the ANCOVA test was performed. Age and gender were used as covariates for all analysis. Linear regression analyses were performed to evaluate correlations between the crunning test and sport frequency, spare time-related aspects, cognitive/neuropsychological aspects. Statistical significance was set for p < 0.05. All estimates were adjusted for gender and age. The software STATA/MP 12.1 (StataCorp. 2011. Stata Statistical Software: Release 12. College Station, TX, USA: StataCorp LP.) was used for the statistical analysis. #### 3. Results #### 3.1. Gender and Age Mean time to perform the crunning test was 6.25 s (SD 2.416, n. = 589). Better crunning performances were observed for males compared to females (independently from age) and for older compared to younger children (independently from gender) (Table 1). The regression coefficient for age was −0.23 (SE 0.051, p = 0.000, n = 589). Table 1. Differences in the crunning test performances by gender and age range. SD: Standard deviation; Tot: Total number; <sup>a</sup> Estimated through paired Student's t-test. <sup>b</sup> Estimated through ANCOVA. All estimates were adjusted for gender and age. #### 3.2. Demographic Factors Table 2 shows the differences in the crunning movement performances by demographic and SES factors. Similar characteristics across participants for each county were observed (Italy, 8.5 ± 1 years; 34.3 ± 9.8 kg; 135.0 ± 9.8 cm; boys, 8.4 ± 1 years; 33.9 ± 9.7 kg; 135.3 ± 9.7 cm and girls, 8.6 ± 1.1 years; 34.8 ± 10.1 kg; 134.7 ± 10 cm; Germany, 8.9 ± 0.8 years; 37.6 ± 9.9 kg; 137.8 ± 7.5 cm; boys, 9.0 ± 0.8 years; 37.6 ± 8.4 kg; 138.6 ± 7.3 cm and girls, 8.7 ± 0.7 years; 37.4 ± 12.7 kg; 136.3 ± 7.9 cm; Portugal, boys, 10.9 ± 1.7 years; 139.1 ± 15.3 cm; 39.4 ± 9.3 kg; Spain, 10.5 ± 1.0 years; 142.7 ± 20.4 cm; 45.1 ± 23.1 kg; boys, 10.4 ± 0.9 years; 145.8 ± 14.1 cm; 43.2 ± 12.4 kg and girls, 10.4 ± 1.0 years; 144.7 ± 13.2 cm; 40.2 ± 10.4 kg; Lithuania, 9.9 ± 1.13 years; 35.7 ± 8.0 kg; 144.0 ± 9.0 cm, boys 9.9 ± 1.19 years; 36.4 ± 8.9 kg; 144.0 ± 9.0 cm and girls 10.1 ± 1.02 years; 34.6 ± 5.8 kg; 142.0 ± 11.0 cm; Croatia 9.4 ± 0.5 years; 35.0 ± 8.2 kg; 138.3 ± 7.6 cm; boys, 9.7 ± 0.5 years; 36.5 ± 7.6 kg; 140.7 ± 7.4 cm and girls, 9.3 ± 0.5 years; 34.7 ± 8.3 kg; 137.8 ± 7.6 cm; and Turkey, girls, 10.8 ± 1.8 years; 134.9 ± 12.7 cm; 45.3 ± 10.9 kg). Significant differences in the crunning movement were found across countries. Table 2. Differences in crunning test performances by country and socio-economic status. SD: Standard deviation; <sup>a</sup> Estimated through ANCOVA. All estimates were adjusted for gender and age. #### 3.3. Sport-Related Aspects Among the sport-related aspects, the following were found to be associated to better performances: team sport type, with children practicing team sports performing better than those practicing individual sports (h/week) (Table 3). Table 3. Differences in crunning test performances by sport-related aspects. SD: Standard deviation; SE: Standard error; <sup>a</sup> Estimated through paired Student's t-test. <sup>b</sup> Estimated through linear regression analysis. \* Five sport categories were included, according to the American Heart Association classification adopted. No differences were found across different sports, current sport frequency, sport type (based on the American Heart Association classification (impact)). #### 3.4. Spare Time-Related Aspects Regarding spare time-related aspects, the following items were correlated to the crunning performances. In general, physical activities such as attending a gym, going to the park or going out with friends were positively associated to performance outcomes (Table 4). Table 4. Differences in crunning test performances by spare time-related aspects. All estimates were adjusted for gender and age. SE: Standard error; <sup>a</sup> Estimated through linear regression analysis. All estimates were adjusted for gender and age. Furthermore, spending more time with parents and siblings seems to be associated with better performances. #### 3.5. Cognitive/Neuropsychological Aspects In general, very low regression coefficients were found. Children with lower school marks had better crunning performances, such relation was present in either children practicing teams and individual sports (Table 5). No significant correlations were found between all the inhibitory control items and the crunning movement for children practicing team or individual sports, with higher times needed to select congruent, incongruent and control trials associated to lower times needed to perform the test. Working memory items were not correlated to the crunning performance. Table 5. Associations between the crunning test performance and cognitive/neuropsychological aspects for team and individual sport practitioners. SE: Standard error; <sup>a</sup> Estimated through linear regression analysis. All estimates were adjusted for gender and age. Items related to the shifting of attention showed positive but very low correlations to crunning times (with higher number of errors and higher time to perform the test correlated to lower crunning performances), with the only exception of the time to recognize numbers and letters in children engaged in team sports, which revealed significant correlation but very low coefficients. #### 4. Discussion With this investigation, we aimed to determine possible factors associated with the performance of the crunning movement. As expected, in our population of European children, males performed better compared to females and older children performed better than younger ones. Such aspects have been reported by various authors across different motor domains. Tomkinson et al. [1] reported for a sample of over 2 million children performances, increases across age groups from 9 to 17 years old, with males performing better than females in terms of strength, power, agility and aerobic capacity. However, the results are in favour of females regarding balance, flexibility and coordination. Similar results are reported by another investigation in a sample of 3804 children ranging between 6 to 10 years of age. Again, boys perform better than girls regarding strength, power, speed, agility and aerobic capacity but not flexibility, with performance increases according to age [32]. Both aspects are consistent with growth and physical maturation [33,34]. #### 4.1. Demographic Aspects The results we obtained regarding the crunning movement performance show differences related to each country with better performances from the Portuguese children and worst performances from the Turkish children. It has to be noted that the Portuguese sample was composed only of male children, while the Turkish sample was composed only of female children. Such results are consistent with the general outcomes provided above. Furthermore, it is not uncommon to appreciate geographical differences when considering different populations of same age ranges, for example, regarding speed in the 20 m sprint between 6-year-old Greek (5.05 ± 1 s) [35] and Lithuanian boys (5.8 ± 1.2 s) [36], or for jumping performance of 10-year-old Colombian girls [20] (110.2 cm), Australian girls (103.25 cm) [37] or South African girls (149.3 cm) [38] in the standing long jump. Differences are also present between the same country when considering rural and city areas [39]. The authors of this latter study indicated that these differences between areas possibly influence individual habits and therefore the level of physical activity which each children experiences. A review by Carlin et al. [40] also analysed the association of environmental factors and levels of PA. The study associated increased levels of PA in neighbourhoods which provided facilities, parks and public equipment, and found the opposite for those with fewer facilities. Therefore, environmental and cultural aspects may possibly explain the difference reported between the test performance of each country within the present investigation. #### 4.2. Sport and Spare Time-Related Aspects In the previous sections, the influence of habits regarding the activity levels was evaluated [41]. The results we obtained in this sample of European children confirm that positive habits and lifestyles are associated with improved performance of the crunning movement. For instance, the better results obtained in those children engaged in gym activities or those going out to the park in their spare time. Our results are in line with a systematic review analysing the relationship between outdoor time and PF in children [42]. The authors report an overall positive effect of outdoor time on PF, however, with no specific effect on musculoskeletal fitness. Another cross-sectional investigation aimed to understand how sedentary behaviours or screen time could affect motor skills in children aged 5–16 years [43]. Screen time in particular was associated with lower physical activity, with greater effects on adolescents compared to children and on girls compared to boys. This aspect, if linked to increased sedentary behaviours, could lead to increased fat mass and therefore decreased PF amongst children [44]. In addition, our results also show that children engaged in team sports performed better than those engaged in individual activities, independently of the activity intensity. Other investigations comparing individual and team sports have reported similar results, for example, Morano et al. [45] evaluated physical and psychological factors among children, finding that children engaged in team activities had better shuttle run results for aerobic performances compared to their peers who were engaged in individual sports, and were also less dissatisfied with their body image. Jukic et al. [46] examined the differences in fundamental motor skills in a sample of under-10 soccer players, indicating that greater motor skills were positively related to gross motor quotient and locomotion skills. School children's enjoyment and cohesion during sport activities can predict physical improvements, and those engaged in team sports show significantly higher levels of enjoyment [47]. Being engaged in team sports has also been associated in children with increased motor skill proficiency [48], with greater associations in boys compared to girls. When neurobiological integrations are analysed, it is possible to see that boys involved in team sports have thinner cortices in the frontal lobe compared to those engaged in individual activities. This aspect indicates a faster maturation of the frontal cortex related to an advantage of frontal areas functioning [49]. These are factors that altogether are determinant to adequately perform a complex skill as the crunning movement, which requires the use of upper and lower limbs simultaneously. #### 4.3. Cognitive and Neuropsychological Aspects Relative to cognitive and neuropsychological aspects, two main findings have been associated to the performance. Firstly, academic achievement is negatively linked to the performance results and, secondly, cognitive function is not linked to crunning performance neither for children practicing team sports nor for those practicing individual sports. Our findings are generally in contrast with other investigations. Emerging literature has positively linked cognitive performance and academic achievement to sport performance [50]. Such associations are also seen in intervention studies where both cognitive functioning and academic achievement are increased following a physical intervention [51–53]. However, the majority of studies have proposed aerobic activity as the main typology of physical activity included within their interventions [54]. These interventions are seen to act by improving brain structure and function [4]. However, within our study, neither a longitudinal intervention was undertaken nor the crunning movement involves aerobic metabolism activation. It is possible that the nature of the crunning movement itself requires a different cognitive demand compared to the cognitive and neuropsychological measures assessed. Limitations of this study are the following: (1) The sample for each country was collected within one school, therefore, it was not possible to evaluate specific local aspects but only broad geographical differences. (2) Not all countries had the availability of both male and female participants. (3) The classification for sport intensity was based on broad characteristics of sporting activities (i.e., soccer, basket, gymnastics, etc.), therefore it is not possible to associate specific aspects related to different approaches between countries. (4) Regarding the cognitive and neuropsychological assessment, not all children and adolescents from the different countries were screened because they were not present at the time of the evaluation. Therefore, the sample which is available for providing information regarding results for such aspects is restricted. Indeed, a broader sample would be required for greater consistency of the test. There is a need to identify the sensitivity of the test to specific physical qualities. Therefore, it will be important in future investigations to compare the present results to other populations in order to verify the applicability of the findings here provided. The present study is the first known study to have evaluated and tried to identify variables associated to the crunning movement. Therefore, it will be necessary to consider gender and physical activity differences among individuals if planning to include the crunning test within a test battery. #### 5. Conclusions The present investigation detected different factors associated with the performance of the crunning movement. These are related to lifestyle and cognitive factors which may influence performance of the crunning movement. These associated variables need to be considered when comparing the results of the crunning movement test, especially across populations. Special attention must be paid regarding gender and previously practiced physical activity. The specificity of the crunning test still needs to be understood within the context of a fitness evaluation. Author Contributions: Conceptualization, A.B.; Data curation, A.B. and F.N. ¸S.; Formal analysis, E.T. and G.T.; Investigation, C.M.d.S., M.B., M.G.-L. and S.P.; Methodology, M.G.-L.; Project administration, M.A. and A.P.; Supervision, M.B.; Visualization, A.R.; Writing—original draft, E.T. and G.T.; Writing—review & editing, A.B., Ö.G. and D.J.S. All authors have read and agreed to the published version of the manuscript. Funding: The work was conducted within the ESA (Enriched Sport Activities) Program—Agreement number 2016-3723/001-001, funded by the Erasmus Plus Sport Programme (2017–2019) of the European Commission. Erasmus+ Sport Programme. EAC/A04/2015—Round 2 E+ SPORT PROJECT: 579661-EPP-1-2016-2-IT-SPO-SCP. Enriched Sport Activities Program. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Lithuanian Sports University's Institutional Review Board, approval No 579661-EPP-1-2016-2-IT-SPO-SCP (2018-02-05). Informed Consent Statement: A parent or legal representative of each child signed a declaration of informed consent. Data Availability Statement: Data available on request due to restrictions eg privacy or ethical. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. #### References #### Article	doab	2025-04-07T03:56:58.866150	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 15 }
0051d55d-461b-414c-a51b-301e45ff4d0c.16	Four Minutes of Sprint Interval Training Had No Acute Effect on Improving Alertness, Mood, and Memory of Female Primary School Children and Secondary School Adolescents: A Randomized Controlled Trial #### Terence Chua 1, Abdul Rashid Aziz <sup>2</sup> and Michael Chia 1,\* Received: 13 November 2020; Accepted: 10 December 2020; Published: 14 December 2020 - Abstract: We investigated whether a 4-min sprint interval training (SIT) protocol had an acute effect (15 min after) on improving alertness, mood, and memory recall in female students. Sixty-three children and 131 adolescents were randomly assigned to either a SIT or control (CON) group by the class Physical Education (PE) teachers. The SIT intervention was delivered twice a week for 3 weeks. SIT participants performed three, 20-s 'all-out' effort sprints interspersed with 60-s intervals of walking while CON group sat down and rested. PE lessons were arranged such that the first two sessions were to familiarise participants with the SIT protocol leading to acute assessments conducted on the third session. On that occasion, both groups rated their alertness and mood on a single-item hedonic scale and underwent an adapted memory recall test. The same assessments were administered to both groups fifteen minutes after delivery of SIT intervention. A 4-min SIT involving three, 20 s 'all-out' effort intensity sprints did not have an acute main effect on improving alertness, mood and, memory recall in female children (η<sup>p</sup> <sup>2</sup> = 0.009) and adolescents (η<sup>p</sup> <sup>2</sup> = 0.012). Students' exercise adherence and feedback from PE teachers are indicatives of the potential scalability of incorporating SIT into PE programmes. Different work-to-rest ratios could be used in future studies. Keywords: sprint-interval training; learning in youths; acute exercise; school; attention #### 1. Introduction Emergent literature suggests that a single session of aerobic exercise has beneficial effects on mood [1] and cognition [2] in adults. Reviews and meta-analyses have found that acute and chronic exercise improves attention and memory in children and adolescents [3,4]. These two domains of cognitive functions are essential for learning [5]. Improved cognition and enhanced mood after an acute bout of aerobic exercise may be explained by psychological and neurophysiological mechanisms. Exercise acts as an arousal stimulus [6]. Synthesis of brain-derived neurotropic factors (BDNF) is up-regulated leading to the activation of a pathway that initiates neuroplasticity and neurogenesis of the hippocampus [7,8]. Increased blood circulation during exercise also promotes more oxygen being delivered to the brain [9]. However, these underlying mechanisms are said to be dependent on exercise intensity. A meta-analysis revealed that studies utilising higher intensity exercise reported greater acute cognitive benefits [2]. The intensity of exercise can also affect mood differently [10]. Therefore, higher intensity exercise may be necessary for any potential positive effect on mood and cognition to be maximised. The acute and chronic exercise effects cannot be considered in isolation. It is explained that exercise training both in the short term (immediate and/or soon after) and long term (days, weeks, months, and years) increases the capacity for exercise, thereby permitting more vigorous and/or more prolonged individual exercise sessions and a more significant acute effect [11]. As such, an acute response to an exercise intervention refers to the transient effects of the exercise immediately or soon after the intervention. Mood, alertness, and memory have facilitative influences on learning. These states of learning are different and but at the same time can be interdependent on each other. Young people experience a range of emotions, from positive emotions like enjoyment to negative emotions like anger, boredom, and anxiety in the process of learning. When negative emotions arise, learning may become a less enjoyable process to them. Consequently, they may be less motivated to interact with teachers and classmates [12,13]. The process of learning involves a continuous effort and awareness to inhibit the shift of attention to irrelevant activities [14,15]. Otherwise, a phenomenon called attentional bottleneck may manifest [16]. Children and adolescents spend six or more hours of their waking time in school, with much of those time spent engaging in sedentary activities due to classroom curriculum requirements and the inactive nature of most post-curriculum school-based activities [17]. Even though a single session of exercise is unlikely to cause impact on mood and cognition lasting throughout the schooling day, it may be opportunistic for qualified physical education teachers to enrich their students' formal learning experience by harvesting the immediate benefits of exercise. The impact of interval training on cognition and learning in young people has generated research interest in recent years. Interval training can be characterized as sprint-interval training (SIT) or high-intensity interval training (HIIT). SIT involves very short bouts of 'all-out' effort sprints while HIIT involves relatively intense but submaximal workloads corresponding to 80–100% of maximal heart rate. Both forms of interval training are interspersed with periods of lower-intensity recovery [18]. Interval training resembles the sporadic patterns of physical play of free-living young people [19,20] and is generally well-tolerated [21,22]. The attractiveness of interval training is that the time invested to complete the exercise is only a fraction of that in traditional endurance training. This time-saving exercise modality is shown to be effective in improving cardiorespiratory fitness and certain cardiovascular and metabolic disease biomarkers in healthy and overweight youths when implemented as an exercise programme over several weeks to a few months [23–25]. Among various interval-training protocols that were studied in the literature, as little as three, 20 s all-out effort cycling sprints performed thrice a week for six weeks has shown to elicit chronic skeletal muscle adaptations linked to increased cardiorespiratory fitness as well as improved cardio-metabolic health biomarkers in adults [26]. This protocol involves a total work duration of 1 min. It is one of the shortest SIT methods to date to demonstrate positive health-related outcomes, albeit in adults. In terms of the acute effect of interval training on cognition in children and adolescents. Previous studies examining the acute effect of interval training on cognition in children and adolescents had used total work durations which were longer than that of Gillen and his colleagues' protocol. Some researchers used shorter intervals of exercise (10–20 s) while others used longer intervals of exercise (30 s) repeated between 8 and 16 times [27–29]. A 15- to 20-s bout of 'all-out' intensity sprint is suggested to be more palatable for children and adolescents rather than sprint bouts of longer durations (e.g., 30 s or 40 s) [30]. Interestingly, a recent study showed that embedding interval-training within the school day has led to increased moderate-intensity physical activity levels in adolescents [31]. Girls are oftentimes less active than boys [32–34] and hence the use of interval-training to increase physical activity has potential implications on them. Besides, girls are an under-served group compared to boys as research evidence in this area is scarce. Therefore, the primary objective of the study was to investigate the acute effect of a three, 20 s 'all-out' effort sprints on self-reported mood and alertness, and memory recall of female children and adolescents when delivered within a physical education (PE) setting. A secondary aim of the study was to gain perspectives from PE teachers about the feasibility of infusing SIT into physical education classes. #### 2. Materials and Methods #### 2.1. Enrolment of Participants and Institutional Ethical Clearance One independent all-girl primary school and secondary school were invited to take part in the study via convenience sampling. The heads of PE department of both schools were contacts of the principal investigator. The school principal gave her consent and permission for conducting the research in schools was granted by the Ministry of Education. The study protocol and ethics was approved by the Institutional Review Board of the university on 4 April 2018 (IRB-2018-02-009-4). Upon discussion with the PE departments, one Primary 5 and one Secondary 1 cohorts were selected for the study. Written child assent and informed consent from the parents were obtained. Sixty-six female children (9–10 years old) and 131 female adolescents (12–13 years old) from the selected cohorts were enrolled in the study. #### 2.2. Study Design The research employed a parallel, randomized control study design with a 1:1 allocation ratio. Participants were randomly assigned to either a SIT or control (CON) group at the individual level by the class PE teachers. The SIT intervention commenced twice a week for 3 weeks (6 sessions) and all PE lessons were conducted in the mornings, between 8 and 10 am. Following at least two separate familiarization sessions, the acute effects of SIT was assessed. Of the thirteen classes involved in the study, a few missed 1–2 SIT sessions due to timetabling constraints as reported by the respective PE teachers (i.e., PE lesson was cancelled when it fell on a public holiday). #### 2.3. SIT Protocol and Familiarisation Sessions Leading to the Day of Acute Assessment The SIT group performed a three-minute warm-up routine (light lower limb stretches) followed by three 20 s 'all-out' effort running shuttle-sprints interspersed with intervals of 60 s of walking. The 3 × 20 s sprint bouts adapted from Gillen and his colleagues' work is one of the shortest SIT protocols to date. A work-to-rest ratio of 1:3 may be appropriate as a study showed that female children and adolescents were able to replicate the peak power generated in the first bout into subsequent bouts of the Wingate Anaerobic Test better than adult women [21]. This suggests that an active rest interval of 60 s derived from the 1:3 ratio may be sufficient for female children and adolescents to re-generate peak anaerobic power. The CON group did not receive the SIT intervention. Instead, they sat down and cheered on the SIT group during the shuttle sprints. Duration of the SIT protocol was four minutes and when warm-up was included, the total exercise session was less than eight minutes. Prior to the study, the qualified PE teachers involved in the research were trained on how to deliver the SIT intervention and acute assessments by the principal investigator and his research assistant. The first two sessions were delivered to familiarise participants what an 'all-out' effort entails in the lead up to the acute assessments conducted on the third session. Each SIT participant was paired with a participant from the CON group who was tasked to count the number of sprint-shuttles completed over a marked out 20-m distance by their partner (data not presented). Upon the teacher's cue, the SIT group sprinted as hard as they could back and forth the marked-out distance for 20 s. Immediately after the 20 s sprint, participants walked to and from the marked-out distance for 60 s. With 15 s to go, participants were instructed to return to the starting position and get ready for the second bout. The process of a 20-s sprint bout followed by a 60-s walking recovery was repeated. Participants then performed their third and final bout of 20 s sprint and recovered right after for 60 s. Throughout the shuttle-sprints, partners of participants from the SIT group counted aloud the number of sprint shuttles completed and cheered the participants on to match or better the number of sprint shuttles completed in the previous bout. Most participants were able to match the number of shuttles completed in the previous sprint (data not reported). Indirectly, this indicated that participants were able to provide a maximal effort throughout the three sprint bouts. #### 2.4. Acute Assessment of Alertness, Mood, and Memory Recall (before and 15 min after SIT) The primary outcome measures (i.e., alertness, mood, and memory recall) were assessed during the 3rd PE lesson. Before the SIT intervention was delivered, participants were asked to rate their mood and alertness from 1–10 on a mood scale [35] and a self-constructed alertness scale (i.e., higher number indicated better mood and greater alertness). The questions posed to them were: 'How are your mood right now? Please circle a number that best represents your current mood.' and 'How alert, watchful or attentive are you right now? Please circle a number that best describes you.' The scale consists of faces with expressions from frown to smiles above the number 1, 5, and 10 in gradations that are intended to reflect a progressive change of feelings. Hedonic scales like these are commonly administered to young children in consumer and food preference research. Although its psychometric properties for assessing mood and alertness are not established, another study has adapted it for the same purpose as the present study [31]. In addition, participants underwent an adapted version of the Rey Auditory Verbal Memory Recall test (RAVRT) [36]. The RAVRT was previously administered to the same age group of participants [37]. Instead of the full five trials, a single trial of recall was used in the present study. The PE teacher read out a list of 15 unrelated nouns each containing two syllables to the participants at a speed of one word per second. After the last word was read out, participants wrote down as many words as they could recall (order and spelling of words were not important). The number of correct words recalled by each participant was recorded. The RAVRT is commonly used in clinical research and practice and has a robust construct validity and internal consistency (Cronbach's alpha coefficient of 0.8) which was found to be closely associated with other tests of verbal learning which renders it to be a valid and reliable psychometric instrument. Fifteen minutes after the SIT intervention was delivered, the same acute assessments were administered but in a different order. A different word list was used in the memory recall test of which the words are what students have learnt before. The delay of 11–20 min after exercise was reported to be the window of opportunity for observing the greatest positive effects on cognition. Positive effects may diminish beyond 20 min whereas assessing too soon after exercise may result in negative effects [2]. The study flow from enrolment of schools to data analysis of acute measures is shown in Figure 1 below. At the end of the study, PE teachers were polled on their perceptions on the feasibility of incorporating SIT-type activities into their PE curriculum (i) to get students fit for sports and (ii) to get students healthy. The PE teachers provided their ratings on a self-constructed 5-point scale, with 1 being 'I do not find it feasible' and 5 being 'I find it very feasible'. To measure overall exercise adherence rate, the average percentage of the number of SIT participants who completed all SIT sessions was divided by the number of SIT participants in each class. #### 2.5. Statistical Analyses SPSS Version 23 (IBM Corp., Armonk, NY, USA) was the statistical tool used. Normality of data and homogeneity of variance within each group was assessed. Missing data was replaced by series mean of each group. A 2 × 2 repeated-measures analysis of variance (ANOVA) was performed to analyse the main intervention effect. Given there were only two levels of measurements of the outcome variables (i.e., before condition and 15 min after condition), the assumption of sphericity was not violated. The measure of effect size was reported as partial eta square, where η<sup>p</sup> <sup>2</sup> = 0.01–0.05 was interpreted as a small effect size, η<sup>p</sup> <sup>2</sup> = 0.06–0.13 was interpreted as a medium effect size and η<sup>p</sup> <sup>2</sup> = 0.14 or greater was interpreted as a large effect size. The level of statistical significance was determined as p < 0.05. Descriptive statistics (mean ± SD) for all outcome variables were reported. Figure 1. Flow diagram through each stage of the parallel, randomized control study design. #### 3. Results #### 3.1. Acute Changes in Self-Reported Alertness and Mood, and Memory Recall Score Separate 2 × 2 repeated-measures ANOVAs were performed to compare the acute effect of a 4-min SIT protocol on improving alertness, mood, and memory recall with the CON group. The results of the univariate analysis of each outcome variable is presented in Table 1 for female children and in Table 2 for female adolescents. Multiple Analysis of Variance (MANOVA) revealed that there was no significant main effect of the SIT protocol on improving all three conditions of learning in female children, F(3, 59) = 0.168, p = 0.918, η<sup>p</sup> <sup>2</sup> = 0.008, as well as in female adolescents, F(3, 127) = 0.528, p = 0.664, ηp <sup>2</sup> = 0.012. The time delay of 15 min following SIT has a significant effect on memory recall, with female children performing better (F(1, 127) = 5.929, p = 0.018, η<sup>p</sup> <sup>2</sup> = 0.089) while female adolescents performed poorer (F(1, 59) = 12.801, p = 0.001, η<sup>p</sup> <sup>2</sup> = 0.09) in the RAVRT after SIT. The pre-to-post-test change in self-reported alertness and mood, and memory recall scores between SIT and CON groups were not significantly different (p > 0.05). These results indicated that a four-minute SIT bout involving a combined one minute of 'all-out' effort sprints had no effect on improving alertness, mood, and memory recall in female children and adolescents. #### 3.2. Exercise Adherence and Teachers' Perceptions on Embedding SIT-Type Activities into the PE Curriculum Most of the female participants (primary school children: 91.8±5.0%, secondary school adolescents: 93.7 ± 9.2%) completed all SIT sessions that were delivered. Common reasons for their absenteeism reported by the class PE teachers were taking sick leave (not related to the exercise) and being out of school for inter-school competitions on the day of PE lesson. Six PE teachers involved in the study rated 4.2 and 4.0 (out of 5) on their beliefs in the feasibility of infusing SIT-type activities in their PE curriculum to get (i) students fit for sport and (ii) to keep students healthy, respectively. None of the participants were reported to have sustained injuries resulting from the 'all-out' intensity sprints under the tutelage of the qualified PE teachers. Table1.Mean±SDofalertness,mood,andrecalloffemaleprimaryschoolchildren. J. Funct. Morphol. Kinesiol. 2020, 5, 92 Interaction between time of assessment and experimental group. Table 2. Mean ± SD of alertness, mood, and memory recall of female secondary school adolescents. \* Interaction between time of assessment and experimental group. #### 4. Discussion The primary objective of the study was to examine the acute effect of a SIT protocol involving three, 20-s 'all-out' effort sprints on improving mood, alertness and memory recall in female primary school children and secondary school adolescents. Of interest was also the PE teachers' perspectives on using SIT in a lesson setting (i.e., teachers' thoughts on infusing SIT-type activity in PE lessons as an intervention to get students fit for sports and keeping students healthy). The key findings of the present study were that a 4-min SIT protocol involving three-, 20 s 'all-out' effort sprint did not have any acute effect on self-reported mood, alertness, and memory recall in female children and adolescents. These results did not support the authors' hypothesis that very brief interval exercise enhances student states of learning. Contrary to the present results, previous studies showed that school-based interval training elicited a positive impact on student alertness. A programme called FUNtervals, a six-minute interval exercise that involved four minutes of dynamic, whole-body exercises such as squats, jumping jacks and running on the spot performed at high-intensity showed acute improvements in selective attention in 88 boys and girls aged 9–11 years [29]. In the cited study, the children made fewer errors in the d2 test, an objective measure of one's selective attention, following the FUNtervals session compared to when they were being assigned to a no-activity break group. This was apparently the only study that examined the effects of HIIT following a brief delay of 11–20 min, as in the present study. It was previously suggested in a meta-analysis that a post-exercise delay of 11–20 min was most likely to elicit positive responses in cognition [2]. Interestingly, a recent study reported that the boost in selective attention in 158 adolescents lasted for an hour after a 16-min HIIT session [38]. The 12- to 16-year-old adolescents in the cited study were instructed to perform 30 s of high-intensity exercise in between rest intervals of 30 s. Findings in the literature on the effect of school-based interval training on mood in children and adolescents are scarce. The SIT protocol in the present study used only one movement task, that is sprinting, rather than a series of different body movements. By the day of acute assessment, the activity became rather mundane to the participants as commented by one of the PE teachers. This could have dampened their motivation and resulted in the lack of change in their self-reported affect. This view concurred with previous findings from Cooper and his colleagues who reported that 10 × 10 s running sprints, interspersed with 50 s of active recovery had no beneficial immediate effect on self-reported energy, tension, and calmness in adolescents [27]. Participants in the cited study reported a higher level of tiredness following the exercise than when they were seated in the resting trial. It is likely that participants' mood was in an attenuated state when the mood questionnaire was administered soon after exercise. These results were in contrast with findings reported by another study [28]. They reported that the mood of 21 adolescents improved significantly following an eight- to 10-min HIIT intervention. The reasons for such mixed results are not readily apparent but differences in interval-training protocols, participant cohorts, and the timings of the assessments are plausible explanations. Few studies have investigated the acute effect of HIIT on memory recall in children or adolescents. Findings in the present study showed negligible effect on memory recall in both primary school children and secondary school adolescents. Similarly, no acute effect on visuo-spatial memory and pictorial memory recall in adolescents were reported in other studies [27,38]. Instead, researchers in the latter cited study showed that selective attention and concentration increased in the second and third hour after the HIIT intervention. It is noteworthy that the HIIT protocol employed in the cited study is four times the duration (16 versus 4 min) of the SIT protocol used in the present study. The interval training protocols used in the cited studies were not identical to that used in the present study. (i.e., work-to-rest ratio; total exercise time). In addition, differing qualitative characteristics of the movement tasks (i.e., cognitive demand and coordinative complexity) may have accounted for the mixed results. It was suggested that activity that requires greater attentional and cognitive resources led to greater extent of improvement in cognition than activities with low cognitive engagement [39,40]. The movement task used in the present study is sprinting which most children and adolescents are quite accustomed to. It also does not require a greater degree of coordination compared to exercises described in other studies. Combining the results of 6 acute studies, the authors of a recent review had found that a single bout of HIIT produced significant yet small to moderate acute effects on executive function and affect in youths [41]. Therefore, whether such brief interval training interventions are useful need to be addressed using different perspectives in different school contexts. For instance, the efficacy of SIT-type programmes that are time-saving and low volume in helping female youths adopt a less sedentary lifestyle outside of school. The use of a single-item hedonic scale is reported elsewhere and is also used for self-reporting purposes in adolescents [28]. Adolescents in the cited study were asked to complete the hedonic mood scale before and after every HIIT session throughout the period of intervention (a total of 24 times). In contrast to the present findings, adolescents' mood following HIIT significantly improved by an average of 0.97. Unlike other questionnaires used in the interval training literature, the single-item hedonic mood scale is not established as a validated instrument. Notwithstanding its unestablished validity, the single-item hedonic scale takes less than one minute to answer and is easily comprehensible to children and adolescents. The total time taken to complete the SIT protocol in the present study is a fraction of the time taken by participants in other studies cited in the literature—i.e., 4 min in present study vs. 10 and 16 min in other studies [27,38]. It is plausible that the exercise dose in the present study was too brief to have any effect on alertness, mood, and memory recall from baseline values (pre-SIT intervention). In the present study, participants reported they were relatively alert and in good mood, and their memory recall scores were not markedly in deficit before the acute SIT intervention. The absence of significant difference is plausibly due to a ceiling effect for improvement [42] since PE sessions were conducted relatively early in the morning of a schooling day where children and adolescents are reasonably rested. #### 4.1. Exercise Adherence and PE Teachers' Perspective on Infusing SIT in PE Classes The qualified PE teachers who conducted the study were specially trained by the principal investigator and his team. To motivate participants to perform 'all-out' intensity efforts, they were encouraged to match or better the number of sprint-shuttles completed in the previous bouts. Although the proportion of participants who managed to match or better their number of sprint-shuttles is not reported in the present study, a majority had completed all SIT sessions conducted (91.8% and 93.7% of the female children and adolescents, respectively). The exercise adherence in the present study compares well with the exercise adherence rate of 90% among Australian adolescents reported elsewhere [43]. The continued participation even after the third session, when acute assessment of alertness, mood and memory recall were administered, is an indication that SIT is an appealing exercise for female children and adolescents in the context of the present study. Additionally, when PE teachers were asked for their perceptions on the feasibility in incorporating SIT-type activities in PE lessons, ratings provided were very encouraging. Like the present study, several studies had situated the delivery of interval training as an exercise intervention during PE classes, albeit for different purposes [44]. On the balance of discussion, it appears that PE classes could be avenues where SIT-type or HIIT-type activities can feature, given its flexibility in incorporating different forms of dynamic exercise movements as well as its time-saving regimen. #### 4.2. Strengths and Limitations of Study A unique contribution of the present study was that it involved a cohort entirely of female participants and was one of the largest cross-sectional study that the authors are aware of. Furthermore, the high retention rate and positive ratings from PE teachers are indications of the potential scalability of introducing SIT-type programme in schools. A limitation of the present study was that the acute assessments for mood, alertness, and memory recall were measured on only one occasion and specifically within the period of 11–20 min (i.e., about 15 min) after the SIT. It is indeterminate in the present study if the prescribed SIT protocol has any abbreviated or transient effect on the aforementioned factors that affects learning outside of the 11- to 20-min window. In addition, as this study was conducted only on female participants, the effect of SIT on male participants remains to be examined. #### 4.3. Future Research Directions Future research could explore different permutations on the work-to-rest ratio of the SIT protocol and examine its acute effect on mood, alertness, and memory recall of children and adolescents in a school-based setting. Interval-training could be embedded during the latter part of a school day when mood, alertness and memory of students are on the wane. These brief exercise breaks should be low in volume so that it does not take up much of class time and they should be curated with participant enjoyment in mind. #### 5. Conclusions A 4-min SIT involving three, 20 s 'all-out' effort intensity sprints had no acute effect on improving mood, alertness and memory in female children and adolescents. The high exercise adherence rate and encouraging ratings by PE teachers are suggestive of the potential scalability of incorporating SIT into PE programme in schools. There is a need for more school-based research to explore the acute effect of different SIT permutations in the context of each school. Author Contributions: T.C.: Research coordination, data collection & analysis, manuscript writing; A.R.A.: Study co-conception, manuscript review and revision; M.C.: Research oversight, study conception, manuscript writing, grant owner. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Institute of Education, Singapore, under the RS-SAA institutional grant (reference no. RS 6/17 MC). Acknowledgments: The authors would like to acknowledge the school principals, teachers in the physical education departments and participants of both schools for their support, involvement and participation in the study. Conflicts of Interest: The authors declare no potential conflict of interest. Availability of data and material: Data used in the manuscript will be deposited to the data repository of the corresponding author's institution. Permission from the corresponding author before accessing the data is required. #### References Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). ## Article The Prevalence of Urinary Incontinence among Adolescent Female Athletes: A Systematic Review *Tamara Rial Rebullido <sup>1</sup> , Cinta Gómez-Tomás 2,\, Avery D. Faigenbaum <sup>3</sup> and Iván Chulvi-Medrano <sup>4</sup> Abstract: This review aimed to synthesize the most up-to-date evidence regarding the prevalence of urinary incontinence (UI) among adolescent female athletes. We conducted a systematic review of studies regarding UI in female athletes less than 19 years of age. This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRIMSA). The electronic databases of PubMed, Embase, Cochrane Central Register of Controlled Trials (CEN-TRAL), Scopus, and Web of Science (WOS) were searched between October and November 2020. After blinded peer evaluation, a total of 215 studies were identified and nine were included. Risk of bias was assessed using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist. This review identified a prevalence of UI in adolescent female athletes between 18% to 80% with an average of 48.58%. The most prevalent sports were trampolining followed by rope skipping. The prevalence of UI among adolescent female athletes practicing impact sports was significantly prevalent. There is a need for further research, education, and targeted interventions for adolescent female athletes with UI. Keywords: pelvic floor dysfunction; women's health; pelvic floor training; youth #### 1. Introduction Urinary incontinence (UI) is defined as any complaint of involuntary loss of urine [1]. Mostly prevalent in women, the broad range of UI is 5–27% [2], with an average prevalence of 27.6% based on a review of population studies [3]. The most common type of UI is stress urinary incontinence (SUI) that is defined as any complaint of involuntary loss of urine on effort or physical exertion [1]. Strenuous exercise has been cited as a risk factor for developing symptoms of SUI [4]. Recently, a subcategory of athletic incontinence was proposed as a new term for a specific SUI that occurs during sport activities or competition [5]. One of the most prevalent pelvic floor dysfunctions reported in female athletes is SUI [6–9]. For instance, a meta-analysis that included 7507 women with age ranges between 12 and 69 years, found that the prevalence of SUI was 33.69% for the female athletes compared to 24.40% in the control group [10]. The younger female athletes seem to display isolated symptoms of pure stress UI which is an uncomplicated SUI without other symptoms of urge incontinence or bladder dysfunction [11]. High-impact sports involving jumping, landing or running have shown the highest prevalence rates of urinary loss among young female athletes [12–15]. A recent meta-analysis by Teixeira et al. found a 35% prevalence rate of UI in female athletes (average age of 23.8 years) practicing different sports. When compared with sedentary Citation:** Rebullido, T.R.; Gómez-Tomás, C.; Faigenbaum, A.D.; Chulvi-Medrano, I. The Prevalence of Urinary Incontinence among Adolescent Female Athletes: A Systematic Review. J. Funct. Morphol. Kinesiol. 2021, 6, 12. https://doi.org/ 10.3390/jfmk6010012 Academic Editor: Cristina Cortis Received: 5 January 2021 Accepted: 24 January 2021 Published: 28 January 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). women, female athletes displayed a 177% higher risk of presenting with UI symptoms [16]. Moreover, female athletes practicing high-intensity activities displayed greater odd ratios of SUI symptoms than those practicing less intense physical activity [9,17]. Similar UI prevalence rates (25.9%) were described in a review with meta-analysis focusing on female athletes involved in high-impact sports such as volleyball, athletics, basketball, cross-country, skiing, and running [8]. UI during practice or competition can cause embarrassment and negatively impact athletic performance. It has been reported that a vast majority of female athletes (~80%) with UI are too embarrassed to tell their coaches, which sustains unawareness of the problem and delays intervention [18,19]. UI can affect an athlete's quality of life and impact performance [20], leading to sport drop-out [15,21]. The underlying mechanisms by which young nulliparous female athletes show higher levels of UI as compared to their sedentary females [16,17] are still not scientifically understood. The continence mechanism during sports practice has been hypothesized to be affected by a variety of kinematic and sport-related factors such as pelvic floor displacement during jumps and running [22,23], neuromuscular fatigue of the pelvic floor muscles during strenuous physical activity [24], and morphological changes of the pelvic floor muscles [25]. Moreover, low energy availability, low body mass index (BMI), estrogen changes, and hypermobility joint syndrome have also been suggested as possible contributing factors for developing UI in female athletes [26,27]. Elite female athletes experiencing UI at an early stage are more likely to report UI symptoms later in life [7]. This is a condition that should be addressed early in life and studied in order to provide better care and support. To date, little is known about the pelvic floor function of young female athletes. Although previous systematic reviews have analyzed the incidence of UI in physically active and athletic females of all ages [4,8,10,16], no previous reports have focused their attention on adolescent female athletes. Given the unique developmental characteristics occurring during adolescence and the previously demonstrated association between high impact training and UI, the prevalence of UI in adolescent athletes needs to be specifically addressed. Our main goal was to identify the prevalence of UI in female athletes less than 19 years of age and provide an understanding of the types of sports associated with the highest prevalence rates. #### 2. Materials and Methods #### 2.1. Information Sources and Search The conduct and reporting of this systematic review complied with the Preferred Reporting Items for Systematic review and Meta-Analyses (PRISMA) guidelines [28]. A systematic search of electronic databases including PubMed, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus, and Web of Science (WOS) was carried out between October and November 2020 independently by two blinded authors. No restrictions on language or publication timeline were applied. The search strategy used keywords, mesh terms, and Boolean connectors (AND/OR) including: "Stress urinary incontinence" OR "urine loss" OR "pelvic floor muscles" AND sport OR athlete OR "female athlete". Search results were limited to species (human) and age (birth–18 years) and source type (journals). #### 2.2. Eligibility Criteria and Study Selection Retrieved titles and abstracts were assessed for eligibility for inclusion, and duplicate entries were removed. The same two authors independently reviewed the text of the studies for eligibility. Articles published up to November 2020 were eligible for inclusion. The criteria for inclusion were: (1) study participants included adolescent females participating in sport or athletic activities; (2) study provides an assessment of UI symptoms; (3) study published in a peer-reviewed journal in any language. Randomized controlled trials (RCTs) with two or more parallel groups and crossover trials, non-RCTs were eligible for inclusion if they met the previously mentioned criteria. The criteria for study exclusion were: (1) participants > 19 years old; (2) participants who underwent any type of pelvic floor surgery; (3) participants during their pregnancy and postpartum period and; (4) systematic review, meta-analysis, or case study. #### 2.3. Data Collection Process and Quality Assessment For each study, data were extracted on the characteristics of the population and intervention such as: (1) last name of the first author; (2) years of publication; (3) study design; (4) sample characteristics (age, sample analyzed, weight, body mass index, sport practice, and hours of weekly training); and (5) instrument assessing symptoms of UI. Risk of bias was assessed independently by two authors using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist [29]. The same two researchers rated the studies and discrepancies were resolved by consensus. Data reporting completeness was assessed by applying the STROBE cross-sectional checklist reporting classified as "not reported or unclear", "some information mentioned but insufficient", or "clear and detailed information provided". #### 3. Results #### 3.1. Study Selection The search strategy yielded 500 potentially relevant studies. After the removal of duplicates, 321 records were screened. Of those, 215 potential titles were selected after the database filter insertion. Among those, only nine studies met the criteria for inclusion and were selected for analysis in this systematic review. The study selection flow chart is shown in Figure 1. Figure 1. Flow diagram for the study selection. #### 3.2. Overview of Study Characteristics Table 1 provides an overview of the characteristics of the studies included in this review. Table 2 provides the participants' characteristics of weight, body mass index (BMI), and hours of training per week. J. ofthestudiesincludedinthis Table 2. Summary of participants' characteristics. \* Mean (IQR); \\ Median (IQR Interquartile range). Our systematic review identified nine studies published between 2002 and 2020. The total sample was 633 female athletes, with an average age of 16.15 years, BMI ranging from 18.9 to 21.7 kg/m2, and 6–19 h of training per week. We calculated a mean of prevalence of 48.58% for all the samples that were involved in different sports. Almost all study designs were cross-sectional (n = 8) where one had a pilot cross-sectional design. The risk of bias was assessed with the STROBE checklist for cross-sectional studies [29]. Figure 2 presents a heat map showing the grading of reporting completeness and quality for selected items according to the Strengthening the Reporting of Observational Studies in Cross-sectional studies. Eighty-seven percent of the articles explained the scientific background and rationale for the investigation and 62% stated specific objectives, including any specified hypotheses. Only 50% of the studies presented key elements of study design early in the paper and described the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection. Study size was only explained in one study [31]. Clarity in defining all outcomes, exposures, predictors, potential confounders, and effect modifiers was applicable for 75% of the studies. Fifty percent of the included studies explained all of the statistical methods, including those used to control for confounding variables. Lastly, all studies summarized key results with reference to study objectives and discussed limitations of the study, taking into account sources of potential bias or imprecision. #### 3.3. Principle Findings This systematic review identified a range of UI prevalence rates ranging from 18.2% to 80% and yielding a mean prevalence of 48.58%. In reports that assessed UI in one specific sport, the highest prevalence rates were found in trampolining (80%) followed by rope skipping (75%) and soccer (62.8%). On the other hand, the lowest rates of UI were found in practitioners of rhythmic gymnastics (31.8%). The main outcome for assessing UI symptoms was the International Consultation on Incontinence short form questionnaire (ICIQ-SF), which was used in 5 of the 9 studies. Only one study used a quantitative measurement of UI through the pad-test [31]. Almost all studies included secondary assessments with questionnaires regarding the impact of UI on quality of life, specific type of urine loss, or associated pelvic floor dysfunctions such as fecal incontinence, sexual dysfunction, and pelvic organ prolapse. Only one study [31] measured muscular strength of the pelvic floor muscles. Of note, two of the included studies assessed female athlete triad risk factors including disordered eating behaviors [19,20]. Two studies assessed athletes' knowledge about pelvic floor muscle training (PFMT) [18,20]. A high percentage of adolescent female athletes (69% to 90%) had never heard of PFMT [18,20]. Moreover, 87% of adolescent female athletes stated they would not mention their UI symptoms to their coach [19]. Figure 2. Assessment of reporting completeness and quality of included studies (STROBE). #### 4. Discussion The aim of the present review was to systematically review the prevalence of UI among adolescent female athletes. Notably, we found a wide range of UI prevalence rates among young female athletes varying from 18% to 80%, with an average prevalence of UI symptoms in female adolescent athletes about 50%. Our results are slightly higher than the meta-analytic data presented by Teixeira et al. [16] for female athletes with an average age of 23.8 years, with a weighted average of 36% of UI prevalence. Additionally, our findings are significantly higher than the study by Hagovska et al. [34] who reported a UI prevalence of 14.3% in 503 adult female athletes (21.1 ± 3.6 years of age) who participated in high-impact sports. Notably, in the aforementioned study, the authors determined the impact of each sport activity based on metabolic intensity rather than on ground impact forces [17,35]. Along these lines, our data are in the range reported by Bø who reported a UI prevalence range between 10% and 55% in female athletes between 15 and 64 years of age [15]. Another review involving female athletes between 12 and 45 years [10], noted average prevalence rates varied from 1% to 42.2% Our review included a total sample of 633 young nulliparous female athletes practicing a wide range of sports. Several studies included samples of athletes practicing different sports. We applied a classification of sport impact based on the study by Groothaussen and Siener [30] that has been specifically applied to the analysis of the impact of sports on the pelvic floor [7,10]. This impact classification is divided in 4 distinct groups: impact grade 3 (>4 times body weight, e.g., jumping); impact grade 2 (2–4 times body weight, e.g., sports involving sprinting activities and rotational movements), impact grade 1 (1–2 times body weight, e.g., such as lifting light weights); and impact grade 0 (<1 time body weight, e.g., swimming). The highest rates of UI in our sample were of grade 3 sports, which included jumping and landing actions (i.e., trampolining and rope skipping). Team sports graded 2 such as soccer, basketball, and track and field were found to display high prevalence rates as well. Impact activities such as running, jumping, and landing have been associated with increased intra-abdominal pressure in the pelvic organs and tissues [22,23]. The additional ground reaction forces placed on the continence structures may lead to displacement or insufficient counteractive muscle activity of the pelvic floor [22]. Another possible mechanism that may explain these prevalence rates is the relatively high metabolic intensity of selected sporting activities that contributes to the possible neuromuscular fatigue displayed by the pelvic floor muscles during training or competition [24]. Overall, the main characteristic of all sports performed in our sample was an impact grade between 2 and 3 [30]. The benefits of sports practice early in life are well established; however, young female athletes are not immune to suffering sport-related injuries or illness [36]. Particularly, the young female athlete can suffer from pelvic floor dysfunctions such as UI as well as pelvic pain and anal incontinence [6,34]. Almeida et al. [34] reported fecal incontinence, dyspareunia, and difficulty emptying the bladder in the female athletic group [34]. Low energy availability in female athletes has been noted as another health impairment that can impair pelvic floor function due to a constellation of hormonal, metabolic, and neuromuscular imbalances [26]. In this sense, Whitney et al. [37] found that female adolescent athletes (aged 15 to 19 years) with low energy availability had a higher prevalence of UI when compared with those with adequate levels of energy. Two studies included in our review assessed for the presence of eating disorders [19,20]. Parmigiano reported that 15% of their sample was at risk for suffering an eating disorder and Gram and Bø noted that 9.3% of adolescent rhythmic gymnasts were at risk for disordered eating [20]. In our review, the average volume of training and BMI of the sample ranged from 18.9 to 21.7 kg/m2 and 6 to 19 h of training per week. Collectively, these observations suggest that the high volume and intensity of training along with low energy availability could be potential risk factors for developing UI in adolescent female athletes. Bø and Sundgot-Borgen described that the presence of UI early in life is a strong predictor for UI later in life (ORR of 8.57) [7]. Moreover, leakage during sport practice has been shown to be a barrier to sports participation for young females [15,21]. Due to the observable health and fitness benefits of sports participation for girls and young women [36,38], additional studies are needed to improve our knowledge regarding pelvic floor dysfunction and implement effective preventative measures in active females. There is a lack of data targeting adolescent females investigating preventative, educational, and treatment modalities for UI. Given the high prevalence of UI in young female athletes and the lack of awareness of evidence-based preventative neuromuscular strategies such as PFMT and pelvic floor therapy [18,20,27], more studies are warranted. Pelvic physiotherapy has been found to be more effective in achieving continence in elite female athletes and pregnant athletes engaged in aerobic exercise compared to non-athletes [27]. For all these reasons, we suggest early screening with specific evaluation tools such as the pre-participation gynecological evaluation of female athletes proposed by Parmiagiano et al. [19] as well as the incorporation of specific neuromuscular training programs for the pelvic floor [13]. Increased awareness and educational programs targeting coaches and all female athletes regarding the pelvic floor musculature and specific dysfunctions such as UI are also warranted. Limitations of this review are the small sample size, heterogeneity, and variability of outcome measures as well as the lack of reliable quantitative outcome measures for UI. The selected studies used validated questionnaires to assess urinary symptoms in young athletes. However, these questionnaires were validated in adult populations. More reliable diagnostic outcomes would improve the quality of the studies. In addition, the analysis of co-founding factors specific to the female adolescent athlete such as menstrual cycle and nutritional status would improve the quality of the studies. We recommend the use of the STROBE checklist for risk of bias study assessment to improve the scientific report of these studies and a classification of sport characteristics and impact, which would additionally improve their comparison and assessment. The development and validation of a specific questionnaire for assessing UI symptoms in adolescent females is warranted. #### 5. Conclusions UI during exercise and sports is a concern for young female athletes. Our findings highlight a 48.8% prevalence rate among adolescent female athletes where practitioners of high-impact sports show the highest prevalence rates. Given the high prevalence of UI among adolescent female athletes involving impact sports graded 2 and 3, concerted efforts are needed to provide early education and implement prevention measures before young female athletes experience the burden of UI. Future research is needed to guide our understanding of the underlying physiopathology and unique characteristics of the adolescent female athlete's pelvic floor muscle activity during impact sports. Author Contributions: Conceptualization, T.R.R. and I.C.-M.; methodology, C.G.-T.; software, C.G.-T.; validation, C.G.-T., I.C.-M. formal analysis, I.C.-M. and C.G.-T.; data curation, C.G.-T.; writing—original draft preparation, T.R.R.; writing—review and editing, T.R.R. and A.D.F.; supervision A.D.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. #### References MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com Journal of Functional Morphology and Kinesiology Editorial Office E-mail: [email protected] www.mdpi.com/journal/jfmk MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel: +41 61 683 77 34 Fax: +41 61 302 89 18	doab	2025-04-07T03:56:58.869821	11-1-2022 14:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0051d55d-461b-414c-a51b-301e45ff4d0c", "url": "https://mdpi.com/books/pdfview/book/4016", "author": "", "title": "Health Promotion in Children and Adolescents through Sport and Physical Activities—2nd Edition", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036511979", "section_idx": 16 }
005998e1-f40a-4c86-b95d-ac77fff26206	# IMPROVING THE NUTRITIONAL CONTENT AND QUALITY OF CROPS: PROMISES, ACHIEVEMENTS, AND FUTURE CHALLENGES EDITED BY : Felipe Klein Ricachenevsky, Marta Wilton Vasconcelos, Huixia Shou, Alexander Arthur Theodore Johnson and Raul Antonio Sperotto PUBLISHED IN : Frontiers in Plant Science #### Frontiers Copyright Statement © Copyright 2007-2019 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. 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For the full conditions see the Conditions for Authors and the Conditions for Website Use. ISSN 1664-8714 ISBN 978-2-88963-018-9 DOI 10.3389/978-2-88963-018-9 ### About Frontiers Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals. ### Frontiers Journal Series The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. 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Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: [email protected] ## IMPROVING THE NUTRITIONAL CONTENT AND QUALITY OF CROPS: PROMISES, ACHIEVEMENTS, AND FUTURE CHALLENGES Topic Editors: Felipe Klein Ricachenevsky, Federal University of Santa Maria, Brazil Marta Wilton Vasconcelos, Universidade Católica Portuguesa, Portugal Huixia Shou, Zhejiang University, China Alexander Arthur Theodore Johnson, The University of Melbourne, Australia Raul Antonio Sperotto, University of Taquari Valley - Univates, Brazil Citation: Ricachenevsky, F. K., Vasconcelos, M. W., Shou, H., Johnson, A. A. T., Sperotto, R. A., eds. (2019). Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-018-9 # Table of Contents 06 Editorial: Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges Felipe Klein Ricachenevsky, Marta Wilton Vasconcelos, Huixia Shou, Alexander Arthur Theodore Johnson and Raul Antonio Sperotto 10 From in planta Function to Vitamin-Rich Food Crops: The ACE of Biofortification Simon Strobbe, Jolien De Lepeleire and Dominique Van Der Straeten 37 Natural Variation in Physiological Responses of Tunisian Hedysarum carnosum Under Iron Deficiency Heithem Ben Abdallah, Hans Jörg Mai, Tarek Slatni, Claudia Fink-Straube, Chedly Abdelly and Petra Bauer Bingbing Luo, Jingguang Chen, Longlong Zhu, Shuhua Liu, Bin Li, Hong Lu, Guoyou Ye, Guohua Xu and Xiaorong Fan 77 Elemental Profiling of Rice FOX Lines Leads to Characterization of a New Zn Plasma Membrane Transporter, OsZIP7 Felipe K. Ricachenevsky, Tracy Punshon, Sichul Lee, Ben Hur N. Oliveira, Thomaz S. Trenz, Felipe dos Santos Maraschin, Maria N. Hindt, John Danku, David E. Salt, Janette P. Fett and Mary Lou Guerinot 89 Genetic Basis and Breeding Perspectives of Grain Iron and Zinc Enrichment in Cereals Ana Luisa Garcia-Oliveira, Subhash Chander, Rodomiro Ortiz, Abebe Menkir and Melaku Gedil Grace Z. H. Tan, Sudipta S. Das Bhowmik, Thi M. L. Hoang, Mohammad R. Karbaschi, Hao Long, Alam Cheng, Julien P. Bonneau, Jesse T. Beasley, Alexander A. T. Johnson, Brett Williams and Sagadevan G. Mundree 132 Biofortification of Cereals With Foliar Selenium and Iodine Could Reduce Hypothyroidism Graham Lyons ### 140 Should Heavy Metals be Monitored in Foods Derived From Soils Fertilized With Animal Waste? Rafael da Rosa Couto, Jucinei J. Comin, Monique Souza, Felipe K. Ricachenevsky, Marcos A. Lana, Luciano C. Gatiboni, Carlos A. Ceretta and Gustavo Brunetto 145 Dynamic Modeling of Silicon Bioavailability, Uptake, Transport, and Accumulation: Applicability in Improving the Nutritional Quality of Tomato Mari C. López-Pérez, Fabián Pérez-Labrada, Lino J. Ramírez-Pérez, Antonio Juárez-Maldonado, América B. Morales-Díaz, Susana González-Morales, Luis R. García-Dávila, Jesús García-Mata and Adalberto Benavides-Mendoza 155 Genetic Basis of Variation in Rice Seed Storage Protein (Albumin, Globulin, Prolamin, and Glutelin) Content Revealed by Genome-Wide Association Analysis Pingli Chen, Zhikang Shen, Luchang Ming, Yibo Li, Wenhan Dan, Guangming Lou, Bo Peng, Bian Wu, Yanhua Li, Da Zhao, Guanjun Gao, Qinglu Zhang, Jinghua Xiao, Xianghua Li, Gongwei Wang and Yuqing He Yu-Min Shi, Chun-Chao Wang, Li-Yan Zhang, Jun-Tao Ma, Ling-Wei Deng, Wan Li, Tian-Tian Xu, Cheng-Zhi Liang, Jian-Long Xu and Zhi-Kang Li 210 Toward Eradication of B-Vitamin Deficiencies: Considerations for Crop Biofortification Simon Strobbe and Dominique Van Der Straeten 229 You Shall not Pass: Root Vacuoles as a Symplastic Checkpoint for Metal Translocation to Shoots and Possible Application to Grain Nutritional Quality Felipe K. Ricachenevsky, Artur T. de Araújo Junior, Janette P. Fett and Raul A. Sperotto Bal R. Singh, Yadu N. Timsina, Ole C. Lind, Simone Cagno and Koen Janssens 254 Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis David Chan-Rodriguez and Elsbeth L. Walker 264 Contribution of NtZIP1-Like to the Regulation of Zn Homeostasis Anna Papierniak, Katarzyna Kozak, Maria Kendziorek, Anna Barabasz, Małgorzata Palusińska, Jerzy Tiuryn, Bohdan Paterczyk, Lorraine E. Williams and Danuta M. Antosiewicz Swati Puranik, Jason Kam, Pranav P. Sahu, Rama Yadav, Rakesh K. Srivastava, Henry Ojulong and Rattan Yadav 385 Genome-wide Identification, Characterization, and Expression Analysis of PHT1 Phosphate Transporters in Wheat Wan Teng, Yan-Yan Zhao, Xue-Qiang Zhao, Xue He, Wen-Ying Ma, Yan Deng, Xin-Ping Chen and Yi-Ping Tong # Editorial: Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges Felipe Klein Ricachenevsky <sup>1</sup> \, Marta Wilton Vasconcelos <sup>2</sup> \, Huixia Shou<sup>3</sup> \, Alexander Arthur Theodore Johnson<sup>4</sup> \ and Raul Antonio Sperotto<sup>5</sup> \* <sup>1</sup> Biology Department, Federal University of Santa Maria, Santa Maria, Brazil, <sup>2</sup> Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal, <sup>3</sup> The State Key Lab of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China, <sup>4</sup> School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia, <sup>5</sup> Graduate Program in Biotechnology, University of Taquari Valley - Univates, Lajeado, Brazil Keywords: nutritional quality, biofortification, plant nutrition, iron, zinc, transporter ### Editorial on the Research Topic ### Edited and reviewed by: Jan Kofod Schjoerring, University of Copenhagen, Denmark #### \Correspondence: Felipe Klein Ricachenevsky [email protected] Marta Wilton Vasconcelos [email protected] Huixia Shou [email protected] Alexander Arthur Theodore Johnson [email protected] Raul Antonio Sperotto [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 08 May 2019 Accepted: 17 May 2019 Published: 06 June 2019 #### Citation: Ricachenevsky FK, Vasconcelos MW, Shou H, Johnson AAT and Sperotto RA (2019) Editorial: Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges. Front. Plant Sci. 10:738. doi: 10.3389/fpls.2019.00738 Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges* Plants are the ultimate source of nutrients for humans and livestock. To date, we use only a handful of species for our subsistence and focus mostly on carbohydrate-rich grains such as wheat, corn and rice. However, these starch-rich cereals are typically nutrient-poor, leaving populations that base their diets on starchy grains with low intakes of many essential nutrients. At the same time, seeds, leaves and roots can accumulate toxic elements depending on species, genotype and growth conditions, representing potential hazards to human health. Improving the nutritional quality of plants, which includes both nutrient enrichment of edible tissues and minimizing the likelihood of contamination, has been a focus of research for years. In this Research Topic a diverse collection of reviews, opinions and original research articles highlight the achievements and future directions in this field. ### BIOFORTIFICATION/NUTRITIONAL QUALITY: AN OVERVIEW Garg et al. review a range of biofortification studies in different crops involving transgenic, conventional, and agronomic approaches and the use of biotechnology, crop breeding, and fertilization, respectively. The authors discuss the limitations of each approach and the challenges that transgenic biofortified crops face regarding consumer acceptance. Taking a different perspective, Capstaff and Miller review the current knowledge and future direction for yield and nutritional quality improvement of forage crops, which have received relatively limited attention compared to cereals, fruits, and vegetables. The authors also discuss the applicability of knowledge obtained from model plants and grain crops coupled with the availability of genomics and bioinformatics to generate improved forage crops for food security. The review by Hameed et al. presents an overview of nutritional improvement efforts in potato (Solanum tuberosum) using both traditional breeding and genetic engineering techniques, including nutrient concentration increase and anti-nutrient decrease. The authors also discuss constraints to potato production, such as biotic and abiotic stresses, post-harvest quality, unfavorable soil and climatic factors. Moreover, genetically modified potato risk assessment/regulation and 6 future breeding techniques using TALENs and CRISPR/Cas9 are discussed. Paul et al. review progress in the Banana21 biofortification program, which aims to increase pro-vitamin A content (and iron in the future) in cooking banana, which is a staple food in Uganda. Vitamin A deficiency is one of the most widespread nutritional problems worldwide, affecting millions of people, and is especially prevalent in children. Importantly, the program includes technology transfer and aims to produce and deregulate transgenic local varieties of banana. Authors provide an overview of the strategies used, to achieve the goal of delivering at least 50% of the estimated average requirement of pro-vitamin A in 300 g of banana per day. Other reviews discuss biofortification for specific nutrients. Strobbe and Van Der Straeten focus on the role of thiamin (B1), pyridoxine (B6), and folates (B9) in plant physiology. Biofortification strategies to enhance B-vitamin in crops using metabolic engineering or breeding are presented. The authors also discuss the concept of multi-biofortification, the simultaneous biofortification of multiple vitamins and minerals, and the possible synergistic or adverse effects of such combinations. Lyons reviews the approaches used for agronomic biofortification of crops with iodine (I) and selenium (Se) in major crops, with a special focus on wheat. Although I is provided by salt iodization, this approach has been shown to be insufficient. The author shows problems derived from combined deficiency in both nutrients, and how current approaches to provide enough I and Se to humans are not sufficient. The review discusses approaches to I and Se agronomic biofortification, comparing results of soil and foliar application of I and Se fertilizers. Finally, Puranik et al. assess recent advances and challenges for Ca biofortification in finger millet (Eleusine coracana), a crop with inherently high grain Ca content, but which needs to be in a bioavailable form to impact human nutrition. As such, integration of a naturally Ca-rich crop like finger millet into global biofortification programs could be a good starting point to alleviate human Ca malnutrition. According to the authors, large-scale protein profiling to identify the complete set of proteins involved in finger millet Ca homeostasis is still unavailable. López-Pérez et al. present a dynamic model to describe absorption, transport, and deposition rates of silicon (Si) in tomato. The model consists of six state equations, using as inputs key environmental factors related to Si absorption and mobilization, such as temperature, pH, CO<sup>2</sup> concentration, and soil organic matter. Use of the model can increase understanding of the agronomic management of Si in plants. On a similar line, da Rosa Couto et al. explore the plants grown in soils with long-term animal waste applications as fertilizers and the potential grain contamination with heavy metals. Although important for both traditional and organic farming, the recurrent use of animal waste can result in changes to soil chemistry that make plants more prone to accumulation of heavy metals, which can harm consumers. The manuscript estimates the potential contamination risks associated with soil concentration of heavy metals after organic waste usage reported in the literature, and calls attention to the need for more careful monitoring to understand how different plant species and their edible parts can become a source of dietary contamination for humans. ### REVIEWS ON FE AND ZN BIOFORTIFICATION Nutritional quality includes many topics, but certainly iron (Fe) and zinc (Zn) biofortification has been one of the most prolific areas in the field. Here we feature updated reviews and opinions focused on these two essential micronutrients that are commonly lacking in human diets. Garcia-Oliveira et al. review the genetic basis of Fe and Zn accumulation in cereals and describe how modern breeding technologies are helping to promote essential element accumulation and bioavailability, while minimizing the accumulation of anti-nutrients such as phytate and hazardous heavy metals that often are transported along with Fe and Zn. The authors discuss how existing genetic variability can contribute to breeding of biofortified cereals, and some of the methodological difficulties in reliably measuring Fe and Zn. Sperotto and Ricachenevsky discuss common bean (Phaseolus vulgaris) biofortification efforts, and how model species lessons can provide shortcuts in finding pathways and candidate genes for Fe, Zn and anti-nutrient manipulation in beans. In particular, the possibility of tissue-specific biofortification of common bean seed coat and cotyledons, as well as decreasing phytate and polyphenols anti-nutrients in the same tissues, is discussed. The mini-review by Ricachenevsky et al. describes the role of root vacuoles in controlling symplastic concentrations of nutrients and toxic trace elements, which in turn affects shoot accumulation. Examples from the literature regarding natural variation in vacuolar transporters and loss-of-function mutants show that the more abundantly an element is stored in root vacuoles, the less it is loaded into the xylem and translocated to shoots and seeds. Thus, manipulation of root storage capacity should be considered in biofortification approaches. In another mini-review, Nozoye highlights the usefulness of increasing nicotianamine (NA) levels in plants, which is achieved by overexpressing nicotianamine synthase (NAS) genes. NA is a metal chelator involved in metal translocation. Plants that accumulate more NA increase Fe and Zn concentrations in edible tissues and can also become more tolerant to Fe deficiency. ### EXPERIMENTAL APPROACHES TO IMPROVE NUTRITIONAL QUALITY Manuscripts in this section describe experimental methodologies important for the improvement of nutritional quality in plants. Due to the high bioavailability of rice seed storage protein (SSP) for human and animal nutrition, increased SSP content is one of the main breeding objectives for improving the nutritional quality of rice. Chen et al. investigate albumin, globulin, prolamin, glutelin, and total SSP contents in milled rice of 527 rice accessions grown in two environments. By associating these nutrient traits with genome sequencing data, they identify novel SNPs and candidate genes related to rice seed protein content and composition which may be useful for future rice molecular breeding strategies aiming at quality improvement. It is well-known that daily consumption of fruits and vegetables lowers the risk of several chronic illnesses, including cardiovascular disease, diabetes, and cancer. The health benefits of vegetable and fruit crops are often attributed to their high content of specific health promoting compounds such as fibers, polyphenols, and vitamins. Glycine is the most abundant free amino acid in horticultural soils and Yang et al. show that exogenous glycine supplementation can increase the accumulation of health-promoting compounds and enhance antioxidant activity in hydroponically grown lettuce. Starch is the main carbohydrate form in wheat grain. Other than the major components of amylose and amylopectin, starch can also interact with minor components such as lipids, proteins, and phosphorus. It is understandable that phosphorus supplies must affect the yield and quality of wheat grain. Zhang et al. employ three levels of phosphorus fertilizer application to wheat fields and find that different levels of phosphorus significantly influence the expression of starch biosynthesis genes, starch synthesis, degradation, and microstructure in wheat grains. The results provide knowledge about the importance of applying appropriate amounts of phosphorus fertilizers for the improvement of wheat yield and starch quality. The high affinity nitrate transporter OsNRT2.1 plays a role in nitrogen uptake and translocation in rice. Luo et al. show that OsNRT2.1 overexpression increases not only nitrate uptake, but also Mn accumulation in rice grains. The reason for high accumulation of Mn by OsNRT2.1 overexpression is probably due to elevated expression of Mn transporter genes, including OsNRAMP3, 5, and 6. This work provides an alternative approach for increasing Mn uptake in plants and could have implications for grain quality. ### EXPERIMENTAL APPROACHES TO FE AND ZN BIOFORTIFICATION We highlight a series of original research papers focused on increasing Fe and Zn accumulation in crops. Díaz-Benito investigate wild-type rice and six transgenic rice lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb) in order to elucidate the role of 2-deoxymugineic acid (DMA) and nicotianamine (NA) on metal distribution in the rice embryo and endosperm. Using a series of approaches the authors conclude that when there are increases in DMA concentration alone or in combination with NA, the prevalent mechanism of seed Fe loading is via Fe(III)-DMA. The study also highlights that a better understanding of transgenic plant phenotypes, using in-depth localized quantification of targeted nutrients, will improve the efficacy of future biofortification strategies. In a similar topic, Tan et al.reports on a strategy for Fe biofortification of chickpea (Cicer arietinum) using genetic engineering. The authors successfully transformed cultivar HatTrick with chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) constitutive expression cassettes. Analysis of NA and Fe levels in the transformed seeds revealed that NA levels were twice those of control lines. The authors suggest that this may have important ramifications in terms of increasing Fe bioavailability in chickpea grains. Zhang et al. examine two sets of backcrossed inbred lines derived from the same donor, and two recipient elite varieties from Southwestern China, to determine the effect of genetic background and environment on grain mineral concentration by QTL mapping. This study allowed confirmation of the results of a genome-wide association study (GWAS) using a set of 698 sequenced accessions, and favorable haplotypes of Fe, Zn, Cd, Mn, Cu, and Se candidate genes were identified. In particular, 37 genes (19.3%) were found to be significantly associated between the QTL targeting traits and the haplotype variations by pairwise comparison, and these genes may be useful for future rice biofortification strategies. Singh et al. tackle the challenge of increasing Zn, Fe and protein concentration in wheat grain by applying exogenous nitrogen (N), Fe, and Zn at different rates and application times. Apart from increased grain yield, relatively higher protein content and Fe/Zn concentration were recorded in wheat grain when a split N application was applied. Furthermore, soil and foliar Fe/Zn supplies combined with a single application of N at sowing increased Zn and Fe concentrations by 46% and 35%, respectively, relative to controls. These results indicate that proper management of N, Fe, and Zn application may could enhance grain protein content and Fe/Zn concentration in wheat. ### FE AND ZN PHYSIOLOGY STUDIES TO PROVIDE TOOLS FOR BIOFORTIFICATION Aiming to develop a new method to quickly characterize rice genes related to metal homeostasis (in particular Fe and Zn), Ricachenevsky et al. investigate the ionome of Arabidopsis Full Length Over-eXpressor (FOX) lines with heterologous expression of rice cDNAs driven by the 35S promoter. The authors identified two lines overexpressing OsZIP7, which had 25% increase in shoot Zn concentrations compared to control. Ricachenevsky et al. found that the gene was able to complement a Zn transport defective yeast mutant, the protein is localized to the plasma membrane, and the seeds of the overexpressing lines had significantly higher Zn concentrations. This technique shows promise toward identifying candidate genes for mineral enhancement. Phytoremediation has frequently been defined as a sustainable bioremediation process that uses various types of plants to remove, transfer, stabilize, and/or destroy heavy metals from the environment in which they grow. In this context, it is important to choose the best "crop for the job". Papiernak et al. suggest tobacco as a possible crop to fulfill this goal. However, despite the interest in the use of tobacco to remove metals from contaminated soil, knowledge of the processes by which tobacco exerts this role is still limited. The authors strived to identify new Zn transport genes in tobacco using an in silico approach, gene expression data, gene cloning and functional analysis in yeast (heterologous expression). The authors conclude that NtZIP1 like is localized at the plasma membrane, in the roots and shoots, and is involved in Zn transport. NtZIP1-like appears responsible for Zn uptake by root cells of the mature basal zone and may be involved in a mechanism to protect the root and leaf cells from accumulating excess Zn. Chan-Rodriguez and Walker perform a genetic dissection of yellow-stripe mutants available in the Maize Genetic Cooperation Stock Center (MGCSC). Grasses are known to rely on Fe(III) chelation for Fe uptake, which involves phytosiderophore secretion and Fe(III)-phytosiderophore complex transport into root cells. Yellow-stripe phenotypes are indicative of Fe deficiency in leaves, and one of the first mutants characterized in maize (ys1) contained mutations in the Yellow-Stripe 1 (YS1) transporter, which is the Fe(III)-phytosiderophore transporter (Curie et al., 2001), while another (ys3) is suggested to have mutations in the phytosiderophore secretion transporter TOM1 (Nozoye et al., 2013). Authors screen 31 yellow-stripe mutants from maize, identifying some allelic to ys1 and ys3, as well as three new, non-allelic mutants which have low Fe levels in shoots and may represent new players in Fe uptake mechanisms of grasses. Abdallah et al. describe a non-model plant species (Hedysarum carnosum), from semi-arid areas in Tunisia, where soils are saline-alkaline and present low Fe availability. Three isolates from H. carnosum were characterized for morphological and physiological responses to low Fe conditions, showing rhizosphere acidification and IRT1-ortholog upregulation, but no Fe reductase activity in roots. Interestingly, one ecotype showed increased Fe deficiency tolerance, which may be linked to more pronounced IRT1 expression under control conditions. This study lays the foundation for using H. carnosum to study adaptation to extreme conditions. ### GENE FAMILY CHARACTERIZATION Two studies provide basic knowledge on important transporter gene families which could be important for further biofortification and/or plant nutrition studies. Qin ### REFERENCES et al. identify 13 NRAMP genes in the soybean genome and document variable expression profiles of GmNRAMP genes among tissues and in response to nutrient stress, suggesting that GmNRAMP proteins perform a range of functions in specific tissues throughout development. Subcellular localization analysis in Arabidopsis protoplasts confirm the tonoplast or plasma membrane localization of these proteins. Teng et al. conduct a genome-wide sequence analysis of PHT1 (phosphate (Pi) transporters) genes in wheat and clone 21 of the genes. The cloned PHT1 genes show Pi-transport activity in yeast cells grown under both low and high Pi conditions. Expression of some TaPHT1 genes at flowering stage positively correlated with P uptake after stem elongation across three P application rates and two wheat varieties, suggesting that modification in PHT1 gene expression may improve P use efficiency under a wide range of P concentrations. ### FINAL COMMENT This Research Topic provides readers with a wide range of manuscripts in plant nutrition and describes novel results relevant to food security and global health. ### AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. ### FUNDING This work was supported by National Funds from FCT - Fundação para a Ciência e a Tecnologia through project UID/Multi/50016/2019. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Ricachenevsky, Vasconcelos, Shou, Johnson and Sperotto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # From in planta Function to Vitamin-Rich Food Crops: The ACE of Biofortification #### Simon Strobbe† , Jolien De Lepeleire† and Dominique Van Der Straeten\* Laboratory of Functional Plant Biology, Department of Biology, Ghent University, Ghent, Belgium Humans are highly dependent on plants to reach their dietary requirements, as plant products contribute both to energy and essential nutrients. For many decades, plant breeders have been able to gradually increase yields of several staple crops, thereby alleviating nutritional needs with varying degrees of success. However, many staple crops such as rice, wheat and corn, although delivering sufficient calories, fail to satisfy micronutrient demands, causing the so called 'hidden hunger.' Biofortification, the process of augmenting nutritional quality of food through the use of agricultural methodologies, is a pivotal asset in the fight against micronutrient malnutrition, mainly due to vitamin and mineral deficiencies. Several technical advances have led to recent breakthroughs. Nutritional genomics has come to fruition based on marker-assisted breeding enabling rapid identification of micronutrient related quantitative trait loci (QTL) in the germplasm of interest. As a complement to these breeding techniques, metabolic engineering approaches, relying on a continuously growing fundamental knowledge of plant metabolism, are able to overcome some of the inevitable pitfalls of breeding. Alteration of micronutrient levels does also require fundamental knowledge about their role and influence on plant growth and development. This review focuses on our knowledge about provitamin A (beta-carotene), vitamin C (ascorbate) and the vitamin E group (tocochromanols). We begin by providing an overview of the functions of these vitamins in planta, followed by highlighting some of the achievements in the nutritional enhancement of food crops via conventional breeding and genetic modification, concluding with an evaluation of the need for such biofortification interventions. The review further elaborates on the vast potential of creating nutritionally enhanced crops through multi-pathway engineering and the synergistic potential of conventional breeding in combination with genetic engineering, including the impact of novel genome editing technologies. Keywords: vitamin metabolism, crop improvement, hidden hunger, malnutrition, plant development, carotenoids, ascorbate, tocochromanols ## INTRODUCTION Ensuring food security to all populations is considered a top priority for global societal progress. Undernourishment has dropped severely in the last decades, from roughly 20% of the world population in 1990 to little above 10% in 2016 (Food and Agriculture Organization [FAOSTAT], 2017). It stands undisputed that continuing efforts should be undertaken to further reduce the ### Edited by: Raul Antonio Sperotto, University of Taquari Valley, Brazil ### Reviewed by: Jorge E. Mayer, Ag RD&IP Consult P/L, Australia Khurram Bashir, RIKEN, Japan ### \Correspondence: Dominique Van Der Straeten [email protected] > †These authors have contributed equally to this work ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 28 September 2018 Accepted: 03 December 2018 Published: 18 December 2018 ### Citation: Strobbe S, De Lepeleire J and Van Der Straeten D (2018) From in planta Function to Vitamin-Rich Food Crops: The ACE of Biofortification. Front. Plant Sci. 9:1862. doi: 10.3389/fpls.2018.01862 10* number of undernourished people in the world, which is still close to 800 million. The successful reduction of malnourishment can partly be attributed to the increase in staple crop yield witnessed over the last decades. Indeed, in the last 25 years, the production per hectare of rice, wheat and potato has risen by 30% (Food and Agriculture Organization [FAOSTAT], 2017). However, these crops often fail to supply adequate amounts of micronutrients, thereby augmenting the prevalence of micronutrient malnutrition (MNM, 'hidden hunger'). These micronutrients include minerals such as iron, zinc, selenium, and manganese, as well as a wide range of vitamins (Miller and Welch, 2013). Hidden hunger affects an alarming two billion people (Bailey et al., 2015; Rautiainen et al., 2016), mostly in the form of anemia, occurring in one-fourth of the human population (McLean et al., 2009). The case of anemia clearly demonstrates the physiological impact of MNM, as its onset has been linked to deficiencies in different micronutrients such as iron, vitamin B1, B9, and B12 (Green, 2003; Imdad and Bhutta, 2012; Stabler, 2013). The importance of MNM is further highlighted by the large, calculated economic benefit a reduction of child malnutrition would have on development. Among 19 prioritized investment-for-development targets listed in the Post-2015 Consensus, the Copenhagen Consensus Center think-tank has ranked the reduction of child malnutrition as the human development investment with the highest potential economic returns (Copenhagen Consensus, 2012). Vitamin deficiencies can be combatted by supplementation, industrial fortification, biofortification, and educational interventions encouraging dietary diversification. It should be noted that choice of the intervention strategy to be implemented depends on regional dietary and cultural differences (Bailey et al., 2015). However, some universally valid remarks can be made. Supplementation, whether by administration of (multi-)vitamin pills or by fortification of cereal products (mandatory in many countries), has shown to be a fast and powerful means to reduce vitamin deficiencies (Sandjaja et al., 2015; Atta et al., 2016; Wang et al., 2016). Unfortunately, this intervention is not easily applicable to poor rural populations in need (Blancquaert et al., 2014). Furthermore, supplementation could exhibit adverse effects, as demonstrated by the observation of increased mortality and higher risk of colorectal cancer in males upon vitamin A and B9 supplementation, respectively (Benn et al., 2015; Cho et al., 2015). Educational efforts, aimed to change the diet and/or processing of food by populations suffering from vitamin deficiencies, are an excellent way to fight MNM, tackling the root causes of the problem. However, these interventions are expensive and imply cultural and agronomical changes, the feasibility of which cannot be guaranteed (Low et al., 2007; Faber and Laurie, 2011). Biofortification, which consists of enhancing the natural vitamin level of food crops, is advocated as a powerful complementary method to fight vitamin malnutrition, circumventing the aforementioned obstructions (Fitzpatrick et al., 2012; Blancquaert et al., 2017; Saltzman et al., 2017). Biofortification of local crops can be considered a sustainable and cost-effective means to reduce vitamin shortage (Meenakshi et al., 2010; De Steur et al., 2015). Two methods of biofortification, apart from agronomical interventions (Cakmak and Kutman, 2017; Watanabe et al., 2017), can be distinguished. First, biofortified crops can be obtained by conventional breeding or using molecular techniques, to obtain novel high-vitamin lines (Ortiz-Monasterio et al., 2007; Bouis and Saltzman, 2017). Unfortunately, this approach relies on the presence of sufficient variation of vitamin levels in sexually compatible germplasm collections (Shimelis and Laing, 2012; Strobbe and Van Der Straeten, 2017). Furthermore, introgression of a certain trait of interest into various region-specific crops demands time-consuming selection over several generations. Novel breeding techniques, however, enable more rapid retrieval of the desired trait via genome wide association mapping (GWAS) or accelerated selection of the introgression lines using marker-assisted breeding (MAB) (Borrill et al., 2014; Esuma et al., 2016). Second, metabolic engineering via GM-technology allows introduction of one or multiple genes of interest, influencing plant metabolism toward increased accumulation of the particular vitamin. As it is not dependent on sexual compatibility of gene source, genetic elements from a very diverse pool could be utilized, including the vast genetic diversity of prokaryotes. Moreover, metabolic engineering can be implemented in a time and tissue-specific manner via selection of promoters with the desired temporal and spatial characteristics. This method, however, demands prior knowledge about specific vitamin metabolism as well as availability of adequate promoters. In principle, it allows the creation of a model vitamin engineering strategy, which can be implemented in a variety of cultivars and crops. However, this cannot be generalized, due to differences in vitamin regulation and metabolism in different crops and tissues (Strobbe and Van Der Straeten, 2017). Interestingly, novel genome editing techniques such as the CRISPR/Cas system allow directed mutagenesis and editing of targeted genomic regions (Cong et al., 2013; Luo et al., 2016), enabling targeted metabolic engineering approaches, though still constrained by the limitation of genetic diversity of the engineered species. A combination of the aforementioned techniques, could offer powerful solutions to alleviate vitamin deficiencies. Biofortification should be carried out with due consideration to its effects on the plant's physiology and not only with the consumers' vitamin needs in mind. The health impact of a biofortified crop could be region specific, due to genetic, environmental and dietary factors. Massive consumption of staple crops with low content of one or more micronutrients appears to be a major factor aggravating the incidence of the deficiency. Therefore, biofortification of these crops is advised. Biofortification endeavors should, however, not solely focus on vitamin content, but take all factors influencing vitamin-specific nutritional value of the particular crop into considerations, such as storage and processing stability, as well as bioavailability (Fitzpatrick et al., 2012; Blancquaert et al., 2015; Diaz-Gomez et al., 2017b). The three vitamins covered in this review–namely vitamin A, C and E–have been the subject of various biofortification approaches due to their impact on human health and very low content in the six major staple crops consumed worldwide (Table 1). But because of their roles in key (g/capita.day)Dataportionconsumptionon the food matrix, can easily amount to 50% each. 1Highest recommended daily allowance (RDA) is depicted (µg/day). 2Retinol activity equivalent. 3Milled equivalent. 4in 2013. enzymatic and stress-related stress response roles, there is a need to bundle the existing knowledge of in planta vitamin metabolism, taking possible detrimental effects on crop growth into consideration. Consequently, proper design of metabolic engineering approaches for vitamin biofortification requires a profound understanding of in planta vitamin biosynthesis as well as its metabolism. In the past decades, major advances have been accomplished in biofortification of different food crops. Fortunately, some of these are already being used to combat MNM. However, the use of metabolically engineered, biofortified crops has not been implemented to date. Interestingly, the imminent commercialization of provitamin A-rich 'Golden Rice' might open doors toward application of other engineered biofortified crops. In this review, the incidence and pathophysiology of the different vitamin deficiencies are discussed, alongside with the status of knowledge on plant vitamin biosynthesis and physiology and the advances made in crop biofortification with these vitamins. ### PROVITAMIN A – CAROTENOIDS Vitamin A is a collective term for different fat-soluble retinoid molecules (Bai et al., 2011), defined as every chemical structure able to fulfill the biological activity of all-trans-retinol (Figure 1C) upon human consumption (Eitenmiller et al., 2016). Carotenoids, comprise over 600 different compounds, only three of which can be metabolically converted to active vitamin-A substances such as retinol (Figure 1) and its oxidized equivalents retinal and retinoic acid (Asson-Batres and Rochette-Egly, 2016). Carotenoids represent the major source of provitamin A in the diet and are present throughout the plant kingdom. The general backbone is formed by head-to-tail linking of eight isoprene units, resulting in a C40-unsaturated chain, lycopene (Figure 1A), a carotenoid precursor (Eitenmiller et al., 2016). The most important carotenoid, β-carotene (Figure 1B), harbors cyclized β-ionone rings on both ends of the C40-chain (Figure 1). Because these molecules consist of long-chain conjugated polyene units, they are sensitive to oxidation, light, heat and acids (Asson-Batres and Rochette-Egly, 2016). Their sensitivity to oxidation, however, enables them to serve as antioxidants in plants and animals, as the radical resulting from interaction with reactive oxygen species (ROS), is much less hazardous by stabilization of the polyene groups. Vitamin A function, however, greatly exceeds its antioxidant properties, as it plays multiple roles in plant and animal physiology. ### Vitamin A Biosynthesis The principal provitamin A for humans is β-carotene, which is composed of two symmetrical retinyl groups. One such retinyl group consists of a retinyl isoprenoid chain and a β-ionone ring which is important for vitamin A action (Figure 1) (Send and Sundholm, 2007). Hence, as α-carotene, γ-carotene and β-cryptoxanthin also carry 1 β-ionone ring, they possess 50% vitamin A activity. Provitamin A is synthesized in plastids in all photosynthetic organisms by enzymes associated fpls-09-01862 December 15, 2018 Time: 15:9 # 3 with the thylakoid membrane, namely phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), lycopene-β-cyclase (β-LCY) and lycopene-ε-cyclase (ε-LCY); or associated in multienzyme complexes (Cunningham and Gantt, 1998). The direct precursor for provitamin A is geranylgeranyl diphosphate (GGPP) (see also vitamin E biosynthesis, 4.1), which is formed by the condensation of the building blocks isopentenyl diphosphate (IPP) and 3 dimethylallyl diphosphate (DMAPP) molecules, by GGPP synthase (GGPPS) (Ruiz-Sola et al., 2016) (Figure 2). IPP is produced in the plastidlocalized 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and DMAPP is its isomerisation product catalyzed by isopentenyl diphosphate isomerase (IDI). GGPP is also the precursor for chlorophylls, ubiquinones, tocopherols, gibberellins and terpenoids (Saini et al., 2015). The first step of the actual provitamin A biosynthetic pathway is the condensation of two GGPP molecules by phytoene synthase (PSY) forming 15 cis-phytoene, assumed to be a rate-limiting step (Fray and Grierson, 1993; Li F.Q. et al., 2008; Cazzonelli and Pogson, 2010). In most plant species multiple redundant PSY genes are present which are differentially regulated. Salt and drought, are environmental factors which induce PSY expression, thereby enhancing carotene levels (Ruiz-Sola et al., 2014; Nisar et al., 2015). Moreover, ethylene is known to have a positive influence on accumulation of carotenoids, inducing PSY expression (Zhang et al., 2018). This aspect is particularly important in fruit ripening and has therefore been studied in mango (Mangifera indica) (Ma et al., 2018), durian (Durio zibethinus) (Wisutiamonkul et al., 2017) and tomato (Solanum lycopersicum) (Su et al., 2015; Cruz et al., 2018). A recent study identified the tomato transcription factor SlCMB1 as a regulator of both ethylene production and carotenoid accumulation (via PSY and PDS) (Zhang et al., 2018). PSY can therefore, in most plants, be considered a master regulator of carotenoid accumulation, given that it is also stimulated by light, directly controlled by transcription factors PHYTOCHROME INTERACTING FACTOR 1 (PIF1) and LONG HYPOCOTYL 5 (HY5) in Arabidopsis photomorphogenesis (Toledo-Ortiz et al., 2010; Llorente et al., 2017). In the subsequent biosynthesis step, directly downstream of PSY, 15-cis-phytoene is transformed into 9,15,9<sup>0</sup> tri-cis-ζ-carotene via a 15,9-di-cis-phytofluene intermediate by two consecutive desaturation reactions catalyzed by phytoene desaturase (PDS) (Pecker et al., 1992; Li et al., 1996; Qin et al., 2007). Subsequently, either a photoisomerization or an isomerization by ζ-carotene isomerase (ZISO) (Pecker et al., 1992; Li et al., 2007) results in 9,9<sup>0</sup> -di-cis-ζ-carotene. Reiteratively, two desaturation reactions are performed by ζ-carotene isomerase (ZDS) producing neurosporene followed by 7,9,7<sup>0</sup> ,90 -tetra-cis-lycopene (prolycopene) (Wong et al., 2004; Dong et al., 2007). Finally, either light or carotene isomerase (CRTISO) isomerizes the cis bonds into all trans-lycopene. This enzyme is a secondary point of regulation, as it is epigenetically regulated via methylation (Cazzonelli et al., 2009). Several cyclization reactions result in the production of bicyclic carotenoids. Lycopene-β-cyclase (β-LCY) catalyzes the addition of β-ionone rings. One β-ionone ring leads to the formation of γ-carotene; a second one forms β-carotene. Lycopene-ε-cyclase (ε-LCY) catalyzes addition of ε-ionone rings, forming δ-carotene. Addition of one β-ionone ring and an ε-ionone ring on the other side of the linear backbone results in production of α-carotene. Essentially, the pathway bifurcates after lycopene synthesis into β,β- and ε,β-carotenoids, and the relative activities of β-CLY and ε-CLY determine the proportion of lycopene funneled to the two branches (Cazzonelli and Pogson, 2010). Hydroxylation of FIGURE 2 \| Provitamin A biosynthesis. Enzymes involved in its biosynthesis are marked in blue. Connections to other vitamin pathways are indicated in red. Filled yellow boxes indicate the external influences on the biosynthesis, affected enzymes surrounded by a yellow square. The regulatory influences on DXS are derived from studies on Arabidopsis (Estevez et al., 2001), those on PSY from studies in maize, rice and tomato (Li F.Q. et al., 2008; Welsch et al., 2008). Cofactors are encircled in gray. Abbreviations (in order of appearance in the pathway): G3P, glyceraldehyde-3-phosphate; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXP, 1-deoxy-D-xylulose-5-phospate; DXR, DXP reductoisomerase; IPP, isopentenyl diphosphate isomerase; IDI, isopentenyl diphosphate isomerase; DMAPP, dimethylallyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotene isomerase; β-LCY, lycopene-β-cyclase; ε-LCY, lycopene-ε-cyclase; BCH1, β-carotene hydroxylase; ZEP1, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; AsA, ascorbate. α-carotene gives rise to lutein, while hydroxylation of β-carotene leads to formation of zeaxanthin. ### Provitamin A Functions in planta Oxygenated carotenoid derivatives are termed xanthophylls, whereas the non-oxygenated analogs are designated as carotenes. Distinct functions are attributed to these two classes of carotenoids. ### Enhancing Light Harvesting and Photoprotection Lipid soluble carotenoids play a major role in photoprotection. The conjugated double bonds in the carbon skeleton function as chromophore, allowing light absorption in the range of 450–570 nm, covering the absorption gap of chlorophyll. Consequently, they function as accessory pigments in photosynthesis, enhancing light harvesting in the blue– green spectral domain (Cogdell and Frank, 1987; Havaux et al., 2004), while also being required for the correct assembly of photosystems (Formaggio et al., 2001). Xanthophylls are crucial in non-photochemical quenching (NPQ) of excess photon energy by thermal dissipation through molecular vibrations (Demmig-Adams and Adams, 1996). The xanthophyll cycle encompasses two antagonistic enzymes, violaxanthin de-epoxidase (VDE) which converts violaxanthin via antheraxanthin into zeaxanthin, and zeaxanthin epoxidase (ZEP) which performs the reversed reactions. This protective mechanism prevents the over-reduction of photosystem II (PSII) and the generation of ROS (Briantais, 1994). When the level of absorbed light exceeds the photochemical capacity of PSII, the acidification of the thylakoid lumen activates VDE. Additionally, ethylene was found to be a negative regulator of the cycle as it influences the activity and activation of VDE (Chen and Gallie, 2015). Overexpression of β-carotene hydroxylase (BCH1), causing a simultaneous increase in zeaxanthin and xanthophyll levels, enhances tolerance to high light and heat stress (Davison et al., 2002). The extra xanthophyll was shown to be associated with the PSII light-harvesting complexes (LHCII), and the plants exhibited reduced leaf necrosis and lipid peroxidation. Carotenes are important to mitigate the generation of ROS during photosynthesis. Carotenoids can quench both triplet chlorophyll (3Chl<sup>∗</sup> ) and singlet oxygen (1O2), protecting PSI and PSII from photoinhibition (Edge et al., 1997; Triantaphylides and Havaux, 2009). On the other hand xanthophylls like zeaxanthin are involved in the protection of the photosynthetic membranes against lipid peroxidation (Havaux and Niyogi, 1999; Davison et al., 2002). ### Stress Signaling Besides their role in photosynthesis, carotenoids perform a function in stress signaling, as stress-imposed singlet oxygen production can lead to a variety of oxidative cleavage carotenoid derivatives, several of which are reactive electrophile species (RES). One example of RES is the volatile β-cyclocitral (β-CC), which is capable of altering <sup>1</sup>O<sup>2</sup> responsive gene expression in relation to stress acclimation (Havaux, 2014). This RESinduced <sup>1</sup>O<sup>2</sup> response could interact with jasmonic acid (JA) signaling and thus compromise the JA-mediated responses to pathogens and herbivores in high light acclimated plants (Ramel et al., 2012). Another example in which carotenoidderived signals are implicated in retrograde signaling resides in the control of chloroplast and leaf development. The albino Arabidopsis (Arabidopsis thaliana) null mutant of ZDS, Arabidopsis zds/chloroplast biogenesis5 (clb5), exhibits abnormal leaf development and cell differentiation with weakened auxin responses. Introduction of the pds3 mutation, compromising ζ-carotene synthesis, rescued the clb5 mutant gene expression and leaf development phenotypes. This suggests that ζ-carotene isomers are implicated in regulating chloroplast biogenesis and leaf development (Avendano-Vazquez et al., 2014). ### Shoot and Root Development Inhibition of carotenoid production disturbs the rhythmic oscillation of the lateral root (LR) clock, necessary for establishment of pre-branch sites (Van Norman et al., 2014). The same decrease in LR capacity was observed when using an inhibitor of carotenoid cleavage dioxygenases (CCDs), but the carotenoid-derived signaling molecule responsible for the influence on root branching remains to be identified (Van Norman et al., 2014). Earlier mutant analysis has revealed the necessity for other carotenoid derived signals in normal development. The bypass1 (bps1) mutant has short roots, a malfunctioning shoot apical meristem and leaf vasculature with an increasing severity in lower temperatures. Grafting experiments suggested the constitutive presence of a mobile root derived 'bypass' signal, which required β-carotene synthesis, but no CCDs. (Van Norman et al., 2004, 2014; Van Norman and Sieburth, 2007). CAROTENOID CHLOROPLAST REGULATORY1 (CCR1) which encodes a histone methyltransferase Set Domain Group8 (SDG8), defines yet another link of carotenoids to shoot development. SDG8 is important for expression of CRTISO (Figure 2). Besides enhanced rosette growth and cauline branching, altered carotenoid content was observed in ccr1 (Cazzonelli et al., 2009). ### Vitamin A in Human Health ### Function and Pathophysiology of Vitamin A Deficiency During the last decades, knowledge on vitamin A functioning in humans has greatly increased, emphasizing its tremendous clinical importance (Wiseman et al., 2017). Retinol and retinal vitamer forms of vitamin A play a pivotal role in proper function of vision and dark adaptation. Human vision depends on the regeneration of the vitamin A derivative 11-cis-retinal, necessary for the formation of rhodopsin (Tang et al., 2013; Hanneken et al., 2017; Tian et al., 2017). Rhodopsin in turn is required as pigment in the retinal receptor responsible for dark adaptation (Sommer, 2008; Wiseman et al., 2017). This explains why vitamin A deficiency (VAD) can lead to xerophthalmia, a pathophysiological condition of impaired vision, starting with night blindness, and ultimately leading to complete blindness due to corneal damage (Sommer, 2008; Chiu et al., 2016). Furthermore, vitamin A is known to have a beneficial impact on innate and adaptive immunity (Lima et al., 2010; Wiseman et al., 2017). Consequently, VAD induces increased susceptibility toward a variety of infections, particularly gastrointestinal conditions (Brown and Noelle, 2015). Anemia, the most prevalent of all micronutrient deficiency-induced disorders, has also been linked to VAD, as vitamin A is able to influence iron metabolism (Semba et al., 1992; West et al., 2007). Human reproduction also depends on vitamin A, more particularly retinoic acid, as it is shown to be necessary in spermatogenesis as well as for proper embryo growth (Hogarth and Griswold, 2010; Clagett-Dame and Knutson, 2011; Wiseman et al., 2017). The above is, however, but a selection of the vast impact of vitamin A in all its vitamer entities on basic human physiology. It also illustrates the urgency of cutting back VAD incidence on a global scale. ### Global Burden of Vitamin A Deficiency Occurrence of VAD can be explained by poor dietary diversification, likely caused by high consumption of staples with low vitamin content (Table 1). An estimated 250 million children of preschool age suffer from VAD (Wiseman et al., 2017; World Health Organization [WHO], 2018). Moreover, 250–500 thousand children develop VAD-induced full blindness each year, half of these cases resulting in death within a year (World Health Organization [WHO], 2018). As VAD is known to have a negative impact on the human immune system (Brown and Noelle, 2015), many infection-related deaths could also, at least partially, be attributed to low vitamin A status, indicating that the incidence of VAD-induced mortality is potentially underestimated. UNICEF reported that vitamin A supplementation programs are able to save 350 thousand children lives annually (Dalmiya and Palmer, 2007; Sommer and Vyas, 2012). Despite these efforts, coverage of the supplementation programs remains poor in many regions, explaining the persistent occurrence of VAD in these populations. Though VAD is much more prevalent in low-income countries (Bailey et al., 2015; Wiseman et al., 2017), there is also a great need to enhance (pro)vitamin A uptake on a global scale, given the existence of VAD-induced disorders in the developed world (Chiu et al., 2016). ### Sources of Vitamin A Provitamin A is present in animal as well as plant derived foods (Bai et al., 2011; Mody, 2017). Meat and dairy products are typically rich in retinyl esters, which can be metabolized to retinol in the human body (Bai et al., 2011). In plant-derived food sources on the other hand, the provitamin A content is represented by carotenoids, β-carotene being the most prevalent (Grune et al., 2010). β-carotene can be converted to retinal by the human β-carotene 15,150–monooxygenase (Lindqvist and Andersson, 2002), which is typically absent in strictly carnivorous mammals. Richly colored fruit and vegetables are good sources of provitamin A. Examples of high provitamin A carotenoid containing crops are carrots, sweet potatoes, pumpkin, kale and spinach (Harrison, 2005). The food matrix within which the vitamin is delivered is also of great importance, as it determines bioavailability, demonstrated by the increasing portion of bioavailable provitamin A in orange juice upon pasteurization (Aschoff et al., 2015). As the provitamin A content of a food source can be the result of a whole array of provitamin A (mostly carotenoids) compounds, the vitamin content is often described as retinol activity equivalent (RAE). The RAE measures the amount of provitamin A expressed as having the same bioactive power of a certain amount of retinal, taking bioavailability into account. The highest recommended daily allowance is 1.3 mg for lactating females. As all major staples, with the exception of plantain (cooking banana, Musa sp.), are poor sources of provitamin A (Table 1), there is a strong case for raising its level in those crops (De Moura et al., 2016). ### Biofortification: Toward Higher Provitamin A Levels in Crops Metabolic Engineering Over the last decades tremendous efforts has been invested in the augmentation of provitamin A levels in different crops (Giuliano, 2017). PSY, responsible for the first committed step of carotenoid biosynthesis, has been pinpointed as rate-limiting step, thereby serving as an ideal candidate gene in biofortification strategies (Fitzpatrick et al., 2012). A well-known example is the genetically engineered Golden Rice (Oryza sativa) (Ye et al., 2000; Beyer et al., 2002; Paine et al., 2005), which has a yellow color, due to its high carotenoid nature. In Golden Rice (Ye et al., 2000), metabolic engineering was achieved via endospermspecific induction of the daffodil (Narcissus pseudonarcissus) PSY and bacterial (Erwinia uredovora) carotene desaturase (CRT), representing the steps in carotenoid synthesis which are naturally not expressed in rice endosperm. The Golden Rice engineering strategy was later improved by replacing the daffodil-derived PSY by a maize ortholog showing a stronger enzymatic activity in rice than the originally used daffodil enzyme and thus leading to higher beta-carotene levels in the so called Golden Rice 2 (GR2) (Paine et al., 2005). The latter rice lines contain up to 3.7 mg/100 g dry weight (DW) carotenoids in the endosperm. GR2 delivers 50% of a child's RDA of provitamin A in 72 g dry rice. On top of its ability to be deployed to minimize VAD, Golden Rice can be considered a solid proof-of-concept, enabling implementation of this metabolic engineering strategy in a range of crops. Indeed, adopting this strategy into Zea mays yielded maize kernels with 6 mg/100 g DW β-carotene (Naqvi et al., 2009), corresponding to a 112-fold increase in total carotenoid content over the WT corn variety used in this study. Also in wheat (Triticum aestivum), this strategy has led to a 10-fold increase in endosperm carotenoid levels, reaching almost 500 µg/100 g dry weight (Cong et al., 2009). Interestingly, a one-gene metabolic engineering approach, overexpressing only PSY, has also led to several successfully biofortified crops. In canola (rapeseed, Brassica napus), PSY introduction yielded 50-fold increase in seed carotenoid content (Shewmaker et al., 1999). Similarly, carotenoid content of potato (Solanum tuberosum) was elevated (up to 3.5 mg/100 g DW) mostly caused by strongly enhanced β-carotene levels (up to 1.1 mg/100 g DW) (Ducreux et al., 2005). In cassava (Manihot esculenta), root specific ectopic expression of PSY resulted in carotenoid levels to be elevated 20-fold, reaching 2.5 mg/100 g DW (Sayre et al., 2011). Finally, a recent cis-genic PSYoverexpression engineering approach resulted in banana lines reaching up to 5.5 mg/100g DW β-carotene equivalent content of fruits (Paul et al., 2017). A different one-gene approach has been applied in tomato fruit engineering (Rosati et al., 2000; Ralley et al., 2016), as this tissue harbors high expression of genes controlling biosynthesis of lycopene, such as the aforementioned PSY. Therefore, a carotenoid biosynthesis gene, downstream of lycopene was a more appropriate choice for biofortification of carotenoid content in tomato fruit (Rosati et al., 2000). The lycopene β-cyclase gene (β-LCY), catalyzing the cyclization of the lycopene molecule by introduction of the β-ionone rings yielding β-carotene (Cunningham et al., 1996) (Figures 1A,B), was engineered in tomato fruit, resulting in high β-carotene tomatoes (Rosati et al., 2000; D'Ambrosio et al., 2004; Ralley et al., 2016). Single gene approaches, despite reaching satisfying levels of provitamin A, could be strengthened by introduction of additional genes, further increasing flux through the biosynthetic pathway. Indeed, further research in canola resulted in seeds with over 1,000-fold increase in β-carotene, reaching over 20 mg/100 g fresh weight (FW) (Fujisawa et al., 2009). This was accomplished by introduction of seven bacterial genes, highlighting the power of multiple gene engineering as well as the applicability of prokaryotic genes (Fujisawa et al., 2009; Bai et al., 2011). Similarly, in potato, combined tuber-specific boosting of PSY, PDS and β-LCY (Figure 2) generated 'golden potato' tubers having 11 mg/100 g DW of carotenoids of which 4.7 mg/100 g DW is represented by β-carotene (Diretto et al., 2007). Another interesting gene in carotenoid biofortification is the gene encoding 1-deoxyxylulose-5-phosphate synthase (DXS). The DXS enzyme acts in the MEP pathway, upstream of IPP formation, in the plastid isoprenoid pathway (Estevez et al., 2001; Sayre et al., 2011; Ruiz-Sola and Rodríguez-Concepción, 2012), thereby acting also upstream of biosynthesis of a whole range of metabolites depending on this pathway, including tocochromanols (see vitamin E). This approach has been adopted in cassava, tomato and Arabidopsis (Estevez et al., 2001; Enfissi et al., 2005; Sayre et al., 2011). The idea of changing carotenoid content via engineering of a further upstream component proves to be applicable, as shown in tomato, as fruit-specific down-regulation of DE-ETIOLATED1 (DET1) [a light signaling pathway controlling gene (Schafer and Bowler, 2002)], leads to enhancement of both carotenoid and flavonoid levels (Davuluri et al., 2005). Fruit-specific RNAi suppression of an epoxycarotenoid deoxygenase (NCED), a key enzyme in abscisic acid (ABA) biosynthesis, resulted in enhanced lycopene and β-carotene levels (Sun et al., 2012). Strikingly, metabolism of different vitamins could be intertwined, potentially positively influencing their accumulation and stability, as was the case with the combined biofortification of vitamin E and carotenoids in 'Golden Sorghum' (Che et al., 2016). This further emphasizes the importance of considering vitamin stability, especially upon long-time storage. In this respect, down-regulation of a lipoxygenase gene (r9-LOX1), known to cause carotenoid oxidation (Wu et al., 1999; Blancquaert et al., 2017) in rice endosperm yielded enhanced provitamin A stability in Golden Rice upon storage (Gayen et al., 2015). Suppressing enzymes involved in vitamin breakdown has also been implemented as a metabolic engineering strategy and successfully demonstrated in wheat. Endosperm-specific stimulation of carotenoid biosynthesis by bacterial phytoene synthase was combined with silencing of carotenoid hydroxylase, leading to kernels accumulating up to 500 µg/100 g DW of β-carotene (Zeng et al., 2015). These strategies are, however, species and likely tissue-specific, as different crops require adjusted engineering approaches. Assessment of their implementation in different agronomically important crops would be a great leap forward (Kang et al., 2017). In this respect, the ability of processing habits to lower vitamin bioavailability should be taken into consideration (Diaz-Gomez et al., 2017a). Interestingly, interventions in provitamin A metabolism resulted in remarkable alterations in crop properties. This has been reported for provitamin A biofortified cassava, achieved by DXS and CTRb (bacterial phytoene synthase) introduction, resulting in prolonged shelf-life upon storage as well as aberrant carbon partitioning causing a significant reduction in dry matter content (Beyene et al., 2018). This further emphasizes the importance of taking all aspects of plant physiology into consideration, not only upon designing but also upon evaluation of biofortified crops. ### Breeding Enhancement of provitamin A content in food crops has not been limited to transgenic metabolic engineering approaches, as different breeding projects have also led to successes (Giuliano, 2017; Haskell et al., 2017). Interestingly, studies implementing genome-wide association (GWAS), association analysis and quantitative trait locus (QTL) mapping, pinpoint the factors strongly influencing carotenoid accumulation. Indeed, as maize exhibits a strong natural variation in carotenoid content, germplasm analysis indicated a lycopene cyclase to be the major determinant of the vitamin level (Harjes et al., 2008). QTL analysis of different crops mostly revealed the same genes to be major effectors in carotenoid accumulation, corresponding to those genes also implemented in successful metabolic engineering approaches such as PSY, LCY, and DXS genes (Giuliano, 2017). Analysis of carotenoid variation could also highlight negative regulators, as was the case for the gene encoding BCH1 (Yan et al., 2010). Molecular techniques have enabled breeding of high vitamin yielding crops. Exemplary cases include biofortified corn (up to 1.5 mg/100 g DW of β-carotene) (Muzhingi et al., 2011; Palmer et al., 2018; Zunjare et al., 2018), cassava (800 µg/100 g DW of β-carotene) (Welsch et al., 2010; Ilona et al., 2017) and sweet potato (400 µg/100 g FW of RAE of provitamin A) (Low et al., 2017). The latter is already reaching almost three million households in Sub-Saharan Africa, thanks to the Sweet Potato for Profit and Health Initiative (SPHI), which aims to provide this orange-fleshed sweet potato (OFSP) to 10 million households (Laurie et al., 2018). Unfortunately, satisfactory variation in rice germplasm to support adequate breeding for enhanced provitamin A content of the endosperm, has not been found (De Moura et al., 2016). A nice overview of achievements in provitamin A biofortified crops is given in a recent review of Giuliano (2017). ## Provitamin A: Major Problems and Future Prospects The successful creation of provitamin A rich rice, coined Golden Rice, is a good example of a product with great potential, the introduction of which is hampered by regulatory obstructions (Potrykus, 2010, 2017). Indeed, though the potential humanitarian benefit as well as adequate cost-effectiveness of Golden Rice are well known (Stein et al., 2006), current societal perception, strongly following the precautionary principle, has blocked the implementation of Golden Rice for almost two decades. Ingo Potrykus, one of the creators of Golden Rice, has referred to this impediment as 'a crime against humanity' (Potrykus, 2010). The rationale behind this, is the calculated amount of Disability-Adjusted Life Years (DALY) (over a million) as well as deaths (over 40 thousand) that could be saved annually by Golden Rice implementation (Potrykus, 2010; De Steur et al., 2017; Wesseler and Zilberman, 2017). The case of Golden Rice holds an important lesson to minimize regulatory obstructions for products of genetic engineering. Satisfactory proof-of-concepts are often difficult to commercialize due to intellectual and tangible property right (Kowalski et al., 2002). When the ultimate goal of a biofortification endeavor goes beyond the academic proof-of-concept, one must thoroughly examine every patent or intellectual property right attached to it. In the case of the Golden Rice project, all licenses -for the technologies involved- have been acquired, enabling free distribution to farmers, provided that the transgenic event is approved (Potrykus, 2017). This was possible, as it is considered a humanitarian project, allowing to be deployed in developing countries by a Humanitarian Use Technology Transfer (HUTT) license. More strikingly, the Golden Rice event GR2-R1 was found to disrupt the native OsAUX1 (encoding an auxin influx transporter) expression, yielding detrimental consequences for plant growth and development (Bollinedi et al., 2017). This further emphasizes the importance of characterizing the genomic place of insertion and potential influences on growth and development. Provitamin A is an example of a micronutrient for which major progress has been achieved in biofortification over the last decades (Giuliano, 2017). A substantial part of these accomplishments has been realized via breeding endeavors (Bouis and Saltzman, 2017; Ilona et al., 2017), without the use of genetic engineering and therefore more readily accepted for commercial release (Potrykus, 2017). Focus should now be directed toward proper information of the public on allowing provitamin A rich crops created via GM-technology, so that these can be deployed to decrease VAD in populations which are in need. Moreover, the case of tomato fruit, which naturally contains sufficient lycopene, thus requiring a downstream metabolic engineering intervention to redirect the biosynthetic pathway, is a nice example on how general knowledge of a food crop steers biofortification approaches. Therefore, acquiring a general metabolic engineering strategy is difficult and future research should first be directed to understanding provitamin A biosynthesis within the target crop tissue as well as natural variation in the germplasm thereof. The latter could put breeding strategies forward as a valuable solution to fight VAD. Finally, given the success of breeding strategies in provitamin A biofortification and the natural variation of sexually compatible germplasm they depend on, expanding the available germplasm of a certain crop could have very beneficial impacts. ## VITAMIN C – ASCORBATE Ascorbate or L-ascorbic acid (AsA), referred to as vitamin C, is a potent water-soluble antioxidant (Iqbal et al., 2004; Macknight et al., 2017). This molecule is, however, unstable, as it easily deteriorates, being sensitive to heat, alkaline environments and oxygen (Iqbal et al., 2004). Vitamin C sensu lato includes all molecules (vitamers) which can be metabolized to form ascorbic acid in human metabolism, including dehydroascorbic acid (Wilson, 2002). Ascorbic acid is a weak sugar acid, related to, and derived from, hexoses (Pohanka et al., 2012). ### Vitamin C Biosynthesis The sole physiologically significant source of AsA is provided via the Smirnoff-Wheeler pathway, following a route via Dmannose (D-Man) and L-galactose (L-Gal), essentially taking place in the cytosol, with the exception of the final mitochondrial step generating L-AsA (Figure 3) (Ishikawa et al., 2008). Therefore, hexoses need to be directed into D-Man metabolism by phosphomannose isomerase (PMI), followed by the conversion of D-Man-6-P into D-Man-1-P by phosphomannomutase (PMM) (Qian et al., 2007; Maruta et al., 2008). The reversible phosphorylation of D-mannose-1-phosphate (D-Man-1-P) by GDP-D-mannose pyrophosphorylase (GMP/VTC1) results in GDP-D-Man, which is subsequently equilibrated with its epimer, GDP-L-galactose (GDP-L-Gal), through GDP-D-mannose-3,5 epimerase (GME) (Wolucka and Van Montagu, 2007). However, this enzyme can also produce GDP-L-gulose, which occurs in 25% of the epimerization events. This leads to the alternative biosynthesis route, named the L-gulose pathway, which might be species or tissue specific. GDP-Gal is converted to L-galactose-1-Phosphate (L-Gal-1-P) by GDP-L-Gal phosphorylase/L-Gal guanylyltransferase (GGP/VTC2), the first committed and ratelimiting step in the vitamin C biosynthesis pathway (Linster and Clarke, 2008). Both transcription and activity of GGP appear light-regulated, explaining the increase in ascorbate levels in high light conditions. Furthermore, as their diurnal pattern of expression was also observed in constant darkness, GGP is assumed to be under circadian clock control (Dowdle et al., 2007) (Ishikawa et al., 2018). Additionally, VTC2 is suggested to be controlled by a cis-acting upstream open reading frame in high ascorbate conditions (Laing et al., 2015). Several other enzymes are also feedback-inhibited by AsA, including PMI (Maruta et al., 2008), GME (Wolucka and Van Montagu, 2003) and LGalDH (Mieda et al., 2004). In the subsequent step in ascorbate biosynthesis, L-Gal-1-P is hydrolyzed to L-galactose (L-Gal) by L-Gal-phosphate phosphatase (GPP/VTC4) (Conklin et al., 2006), followed by an NAD-dependent oxidation into L-galactono-1,4 lactone (GalL) by L-galactose dehydrogenase (L-GalDH). The last step is yet another oxidation, exerted in mitochondria by the flavin containing L-galactono-1,4-lactone dehydrogenase (L-GalLDH), forming AsA which uses cytochrome c as an electron acceptor (Wheeler et al., 1998; Leferink et al., 2008). This enzyme also shows a diurnal expression pattern (Tamaoki et al., 2003). In the case of the alternative gulose pathway, L-gulono-1,4-lactone is formed, and further converted into AsA by L-GulL dehydrogenase (Wolucka and Van Montagu, 2003). Both GalL and AsA, being low molecular-weight solutes, might cross the outer membrane without the need of a carrier. ### Vitamin C Functions in planta The physiologically active form of vitamin C is its anionic form, ascorbate. The water soluble ascorbate anion (AH−) is a universal player in both enzymatic and non-enzymatic antioxidant defense systems and therefore implicated in a range of processes in plants. Its efficiency as an antioxidant most probably relies on the (relative) stability of its primary oxidation product, the monodehydroascorbate radical (MDA) and moreover, on its capacity to terminate radical chain reactions by spontaneously disproportionating into the non-toxic, nonradical product AsA and dehydroascorbate (DHA) (Noshi et al., 2016). ### Antioxidant AsA is of great importance during photosynthesis, firstly because it is capable to donate electrons to PSI and PSII in both normal and stress conditions (Mano et al., 2004; Ivanov, 2014). Moreover it eliminates directly superoxide (O<sup>−</sup> 2), hydroxyl radicals (•OH) and singlet oxygen (1O2) coming from photoreduction and photorespiration and aids in the scavenging of hydrogen peroxide being a cofactor of ascorbate peroxidase (APX) in the Asada-Halliwel pathway or Mehler-peroxidase pathway (Foyer and Halliwell, 1976; Shigeoka et al., 2002). The latter pathway, also known as the ascorbate-glutathion cycle (ASC-GSH cycle), involves APX, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and is of uttermost importance in the antioxidant defense of plants (Foyer and Halliwell, 1976). Despite the multiple scavenging processes present in plants, lipids still receive the burden of oxidative stress leading to the generation of lipid peroxyl radicals. Clearing thereof is accomplished by α-tocopherols (see vitamin E), which in turn are recycled through the oxidative action of AsA (Davey et al., 2000). In addition, ascorbate, being the cofactor of violaxanthin de-epoxidase (VDE), plays a role in the xanthophyll cycle, as mentioned above in the section of vitamin A, protecting PSII from photoinhibition (Eskling et al., 1997). ### Development A wide range of hormone-AsA interactions influence plant physiology. First, AsA is involved as a cofactor of GA3-oxidase and ACC-oxidase in the biosynthesis of gibberellin (GA) and ethylene, respectively (Arrigoni and De Tullio, 2000; Van de Poel and Van Der Straeten, 2014). Second, hormones can also control AsA biosynthesis. In seed tissue, enhanced levels of ABA suppresses activity of NADPH oxidases, the main producers of ROS in seeds (Ishibashi et al., 2017). The resulting decrease of ROS in the aleuron layers inhibits AsA and concomitant GA biosynthesis (Ye et al., 2012). On the other hand, ROS, and more specifically exogenous H2O2, were shown to enhance expression in imbibed seeds of biosynthesis genes of GA, an essential hormone in seed germination (GA20ox1, GA20ox2, GA20ox3, GA3ox1, and GA3ox2) (Liu et al., 2010; Ye et al., 2012). Furthermore, AsA was shown to be implicated in sustaining seedling growth. Simultaneous loss of function of two homologs (vtc2-1 and vtc5-1 or vtc5-2) encoding the biosynthesis enzyme GGP, results in growth inhibition after cotyledon expansion, followed by bleaching. In later stages of development, ascorbate is required for growth, as the older leaves of the rescued double mutants started to bleach again when transferred back to L-Gal-free medium, the immediate downstream product of these isoforms. Moreover, growth reduction was already observed in the vtc2 null mutant, in accordance with its low ascorbate level (20%) as compared to wild-type (Dowdle et al., 2007). AsA is also linked with cell expansion and division. Culture experiments showed an increase in ascorbate levels during cell elongation in tobacco, while addition of an ascorbate biosynthesis inhibitor (lycorin) induced cell cycle arrest in G1 in onion root cells (Liso et al., 1984; Kato and Esaka, 1999). This link could partially be attributed to its function as a cofactor of prolyl hydroxylase which converts proline residues in hydroxyprolinerich glycoproteins such as extensins in the cell wall (Fry, 1986; Kerk and Feldman, 1995; De Tullio et al., 1999; Joo et al., 2001; Sanmartin et al., 2007). Moreover, the observation of a depleted level of ascorbate together with an increased activity of ascorbate oxidase (AOX) in the quiescent center (QC) in maize roots are suggestive for a role of ascorbate in the maintenance of QC identity. The concomitant augmented auxin level revealed a regulatory role of the latter on AOX expression (Kerk and Feldman, 1995). Moreover, shoot apical dominance is stimulated by ascorbate (Barth et al., 2006; Kotchoni et al., 2009; Zhang C.J. et al., 2011). Through control of GA and ABA, AsA is also involved in flowering, programmed cell death and senescence (Barth et al., 2006; Kotchoni et al., 2009). AsA and ABA were also shown to influence the expression of senescence associated genes (SAGs) in an antagonistic way (Barth et al., 2006). Finally, fruit ripening is also related to AsA (Sanmartin et al., 2007). Ascorbate aids in fruit ripening by its counterintuitive site-specific pro-oxidant function. This involves the apoplastic conversion of O<sup>2</sup> and Cu2<sup>+</sup> into H2O<sup>2</sup> and Cu+, which thereupon combine to generate OH radicals. The presence of the latter results in polysaccharide degradation causing fruit softening (Fry, 1998). In addition, AsA is involved in ethylene biosynthesis (see above), which is essential to induce ripening, and in turn, induces AsA biosynthesis via upregulation of VTC4 expression (Figure 3) (Ioannidi et al., 2009). FIGURE 3 \| Biosynthesis of vitamin C. The enzymes committed to vitamin C (VTC) biosynthesis are marked in blue. Feedback regulations are illustrated in purple. Filled yellow boxes indicate the external influences on the biosynthesis, regulating enzymes surrounded by a yellow square. Biosynthesis and salvage links to vitamins A and E, in dark red, are indicated with a double and a dashed arrow, respectively. The dashed oval arrow represents the recycling of α-tocopherol, within which ascorbate aids in the detoxification of tocopheroxyl radicals. Abbreviations (in order of appearance in the pathway): PMI, phosphomannose isomerase; PMM, phosphomannose mutase; VTC1/GMP, GDP-D-mannose pyrophosphorylase; GDP-D-mannose, guanosine diphosphate mannose; GME, GDP-mannose-3<sup>0</sup> ,50 -epimerase; VTC2/GPP, GDP-L-galactose-phosphorylase/L-galactose guanylyltransferase; VTC4/GPP, L-galactose 1-phosphate phosphatase; L-GalDH, L-galactose dehydrogenase; L-GalLDH, L-galactono-1,4-lactone dehydrogenase; L-GulLDH, L-gulono-1,4-lactone dehydrogenase. ### Vitamin C in Human Health fpls-09-01862 December 15, 2018 Time: 15:9 # 12 ### Functions and Pathophysiology of the Deficiency In human physiology, ascorbate functions as an important scavenger of ROS, such as hydrogen peroxide (Lobo et al., 2010). Importantly, ascorbate is also required as a reducing agent in the conversion of iron from ferric (Fe3+) to ferrous (Fe2+) oxidation state, thereby aiding in sufficient iron uptake and thus indirectly linked to anemia in case of deficiency (Iqbal et al., 2004; Macknight et al., 2017). Furthermore, vitamin C assists in the metabolism of tryptophan, tyrosine and folate (Iqbal et al., 2004). Moreover, AsA aids in lowering excess cholesterol levels, thereby reducing atherosclerosis (Das et al., 2006; Chambial et al., 2013). This vitamin is also known to function as a cofactor in several reactions such as hydroxylation of muscle carnitine, amidation of several hormones, and the conversion of the neurotransmitter dopamine into norepinephrine (Chambial et al., 2013). Hence, the function of ascorbate is evidently linked to energy metabolism. In collagen biosynthesis, prolyl and lysyl hydroxylases utilize AsA as a enzymatic cofactor (Myllylä et al., 1984; Pimentel, 2003). This explains the pathogenesis of scurvy, a vicious disease, caused by severe vitamin C deficiency, characterized by bleeding gums and eventually leading to edema, jaundice, hemolysis, spontaneous bleeding, neuropathy and death (Leger, 2008). Strikingly, there have been indications that ascorbate supplementation could have a negative impact on tumor development (Cha et al., 2013; Mastrangelo et al., 2018). Moreover, high vitamin C status could prevent or cure several infections (Carr and Maggini, 2017). Evidence indicates that low vitamin C status, though not immediately depicting clinical symptoms, hampers ideal human functioning, as increasing vitamin C uptake is known to be beneficial (Johnston et al., 2006, 2014). ### Prevalence of Vitamin C Deficiency Incidence of Vitamin C deficiency is difficult to quantify, as clear deficiency-induced disorders only occur upon very severe ascorbate shortage. Furthermore, there is no consensus on ideal vitamin C intake quantities (Frei et al., 2012; Hickey et al., 2014). Indeed, retrieving an ideal recommended daily intake for vitamin C has been a heavily debated issue, even tackled by Nobel Prize winner Linus Pauling (Pauling, 1974). However, it remains undeniable that increasing the vitamin C status would exhibit positive effects on general human health (Macknight et al., 2017). There is, however, no controversy about the presence of vitamin C deficiency in the general public, despite the infrequency of scurvy. Vitamin C status was reported as being deficient in about 20% of the low-income population of the United Kingdom (Mosdol et al., 2008). Comparable results were obtained by analysis of the north-American population, where smoking and low socio-economic status were identified as risk factors for vitamin C deficiency (Cahill et al., 2009; Schleicher et al., 2009). ### Vitamin C Sources Most animals are capable of de novo ascorbic acid biosynthesis, given its vital role in their metabolism. However, humans (but also guinea pigs and bats) have lost this privilege due to mutation in the L-gulono-γ-lactone oxidase (GLO) gene (cf. L-GulLDH in Figure 3) (Nishikimi et al., 1988; Imai et al., 1998), the evolutionary reason of which has been questioned (De Tullio, 2010). This leaves humans dependent on sufficient dietary ascorbate intake to preserve vital functioning. Fresh (citrus) fruits, tomatoes, broccoli and leafy vegetables are considered excellent sources of vitamin C (Iqbal et al., 2004; Chambial et al., 2013). Unfortunately, ascorbate is prone to deteriorate upon storage or processing, as its content declines upon exposure to heat and oxygen (Lee and Kader, 2000). Vitamin C losses during storage can be decreased via limited exposure to heat and oxygen (Lee and Kader, 2000; Sapei and Hwa, 2014). Though most staples are poor sources of vitamin C, potato and cassava do supply a significant amount of the vitamin to the populations relying on these crop products (Table 1). However, elevating the levels of vitamin C in these crops could deliver additional health advantages. ### Vitamin C Biofortification Metabolic Engineering Metabolic engineering strategies, aimed at elevating ascorbate levels in a specific crop/tissue, have been deployed by increasing either ascorbate biosynthesis, salvage or altered pathway regulation (Macknight et al., 2017). Interestingly, these approaches possess the ability to increase tolerance to abiotic stresses such as drought, salinity, cold, heat and high light. In ascorbate biosynthesis, the conversion of GDP-L-galactose to L-galactose-1-P, the central step in plant ascorbic acid biosynthesis, carried out by the GDP-L-galactose phosphorylase (GGP, VTC2) enzyme (see Figure 3), is mainly considered as being rate-limiting, thereby a prime target for metabolic engineering approaches (Bulley and Laing, 2016; Macknight et al., 2017). This has been adequately demonstrated in tomato and potato, where introduction of the kiwi and potato GGP gene, respectively, yielded an ascorbate increase up to sixfold in tomato fruit and threefold in potato tubers (Bulley et al., 2012). Though other steps in ascorbate biosynthesis have been evaluated in metabolic engineering, GGP remains the most successful (Macknight et al., 2017). Ascorbate salvage on the other hand, the retrieval of ascorbic acid from the oxidized dehydroascorbic acid vitamer, has been tackled using dehydroascorbate reductase (DHAR) (Li et al., 2012). Similarly, ascorbate degradation has been engineered via RNAi-mediated downregulation of AOX in tomato fruit, resulting in augmented vitamin C levels (Zhang Y.Y. et al., 2011). Furthermore, the Arabidopsis ethylene response factor AtERF98, positively regulating ascorbate biosynthesis, has been implemented in metabolic engineering attempts, as its overexpression in Arabidopsis resulted in enhanced ascorbate levels concomitant with increased salt tolerance (Zhang et al., 2012). This should, however, be approached with caution, as the impact on other aspects of plant metabolism/physiology requires in-depth knowledge of the affected metabolic pathways (Macknight et al., 2017). Moreover, AsA stability should be considered upon evaluation of metabolic engineering strategies. Indeed, after 8 months storage, a drop of vitamin C levels of almost 90% was demonstrated in pasteurized pink guava nectar juice (Psidium Strobbe et al. Vitamins A, C, E Enhancement in Crops guajava L.) (Ordonez-Santos and Vazquez-Riascos, 2010). Thus, a metabolic engineering approach combining multiple aspects of ascorbate metabolism including as biosynthesis, recycling, stability and potentially regulation, might prove to yield higher but also stable vitamin C augmentation. ### Breeding Given the relatively low increase in ascorbate levels upon metabolic engineering approaches, breeding methods might catch up with these interventions. In pepper (Capsicum annuum), which can be considered a rich source of vitamin C, a 2.5 fold variation was observed within the 7 genotypes examined (Geleta and Labuschagne, 2006). The high heritability of this trait indicates a great potential in breeding programs in vitamin C biofortification of pepper. In tomato, transcriptomic analysis of an introgression line exhibiting 4-fold difference in fruit AsA content, pinpointed pectine degradation (particularly pectinesterases) as an important determinant for vitamin C accumulation (Di Matteo et al., 2010; Ruggieri et al., 2015). By QTL mapping of introgression lines, tomato fruit ascorbate levels were also linked to a single nucleotide polymorphism (SNP) near the MDHAR genomic region (Sauvage et al., 2014; Bulley and Laing, 2016). Subsequently, analysis of a high ascorbate/carotenoid introgression line enabled identification of an L-ASCORBATE OXIDASE allele (AOX) as a determinant for AsA levels, the expression of which negatively correlated with vitamin C content (Calafiore et al., 2016). Interestingly, the same study identified an NCED allele, to indirectly control AsA accumulation. In apple, a sixfold variation in AsA content found over 28 commercial varieties allowed creation of a mapping population, pinpointing GGP alleles as major determinants of fruit vitamin C content (Mellidou et al., 2012). Together, these findings illustrate the vast potential of screening crop germplasms for high vitamin C accumulating varieties, and implement these plants in GWAS and breeding programs. ### Ascorbate: Major Problems and Future Prospects Given its antioxidant nature and a diversity of potential roles, pathophysiological manifestations are not easily attributable to ascorbate deficiency. This is likely the main cause for the dissent on the ascorbate RDA value, which in turn provokes an underestimation of vitamin C deficiency. Therefore, there is a great need to further underline the tremendous health benefit of improving ascorbate status on a global scale, despite the absence of typical deficiency symptoms. As inherent ascorbate levels in wheat and rice endosperm are negligible (Table 1), metabolic engineering strategies in these tissues might be challenging. However, ascorbate metabolic engineering strategies could be fruitful in helping these crops cope with abiotic stresses. Moreover, metabolic engineering has the potential to convert potato into an ideal medium to deliver sufficient quantities of a potent water-soluble antioxidant, ascorbate, to the population. Future biofortification strategies on the other hand, should, based on the available knowledge on ascorbate function in plant physiology, try to exploit ascorbate accumulation to enable creation of nutritionally enhanced crops with concomitant increased stress tolerance. ## VITAMIN E – TOCOCHROMANOLS Vitamin E or tocochromanols, which includes tocopherols and tocotrienols, are fat-soluble, amphipathic molecules (Colombo, 2010). These molecules consist of a lipophilic isoprenoid chain carrying a polar chromanol ring, providing their amphipathic nature (Figure 4). The molecular structure of these vitamers contains three chiral centers, resulting in 8 stereoisomers of each vitamin E entity (Figure 4). Depending on the substituents on the chromanol ring, both tocochromanols groups exist as α-, β-, γ-, and δ-isomers. Vitamin E molecules are known as potent antioxidants, as they are free radical scavengers, of which α-vitamers are most powerful (Niki and Traber, 2012). ### Vitamin E Biosynthesis Tocochromanols are synthesized only in the plastids of photosynthetic organisms. While tocopherols are present throughout the plant, tocotrienol is found almost exclusively in seeds and fruits. Both groups and their isoforms occur in different tissues and exert different functions. α-tocopherol resides mainly in the leaves of vascular plants, while γ-tocopherol is the predominant form in seeds (Grusak and DellaPenna, 1999; Abbasi et al., 2007). Indeed, as seen in Arabidopsis, seeds typically exhibit a more pronounced γ-tocopherol contribution to the total tocopherol pool (Gilliland et al., 2006). The precursors of tocochromanols are derived from two different pathways, the shikimate and the MEP pathway, which are also delivering the precursors of the plastidial biosynthesis of folate (B9) and carotenoids (provitamin A), respectively (Mène-Saffrané and Pellaud, 2017). The polar phenolic p-hydroxyphenylpyruvic acid (HPP), synthesized from tyrosine by tyrosine aminotransferase (TAT) and therefore the shikimate pathway (Figure 5), is used to produce the aromatic ring of the tocochromanols (Figure 4). HPP dioxygenase (HPPD) catalyzes the onset of the actual tocochromanol biosynthesis by converting HPP into homogentisic acid (HGA) after which the pathway bifurcates toward the production of tocopherols and tocotrienols through condensation of two different metabolites bearing the polyprenyl chains (Figure 5). The MEP pathway delivers the precursors for the biosynthesis of prenyl side chains of tocochromanols, as described for provitamin A biosynthesis (see section "Provitamin A Functions in planta"). This branch of tocochromanol biosynthesis utilizes GGPP (geranylgeranyl diphosphate). Interestingly, this product serves as a substrate of multiple enzymes in biosynthesis of different metabolites, including carotenoids, gibberellins, and plastoquinones (Ruiz-Sola et al., 2016). Reduction of GGPP by geranylgeranyldiphosphate reductase (GGDR) yields phytyl diphosphate (PPP) (Gramegna et al., 2018). In the absence of light, PIF3 physically interacts with the promoter of GGDR, down-regulating its expression. Light activation of phytochromes prevents that interaction, leading to transcriptional derepression of the GGDR promotor. The resulting product of GGDR activity, PPP, can be utilized for both tocopherol and chlorophyll biosynthesis (Tanaka et al., 1999). Moreover, PPP is recycled from chlorophyll breakdown, by phytol kinase (VTE5) and phytolphosphate kinase (VTE6) (Vom Dorp et al., 2015). This was revealed by feeding studies in Arabidopsis which demonstrated the incorporation of labeled phytol in tocopherols in seedlings (Ischebeck et al., 2006). Notably, in ripening fruit tissues, often an important source of tocochromanols, recycling of phytol from chlorophyll breakdown is witnessed to be the predominant PPP source (Gramegna et al., 2018). Condensation of PPP and HGA by HGA phytyl transferase (HPT/VTE2) leads to the formation of 2-methyl-6-phytylbenzoquinol (MPBQ), a step toward creation of tocopherols (Sattler et al., 2004). On the other hand, HGA geranylgeranyl transferase (HGGT) catalyzes the condensation of GGPP with HGA, yielding 6-geranylgeranyl-benzoquinol (MGGBQ), leading toward the formation of tocotrienols (Cahoon et al., 2003; Mène-Saffrané and Pellaud, 2017). These two benzoquinol products, MPBQ and MGGBQ, resulting from HGGT and MGGBQ action, giving rise to tocopherols and tocotrienols, respectively, mark the branch point of tocopherol/tocotrienol biosynthesis. This is illustrated by higher accumulation of tocotrienols in HGGT-overexpressing barley (Hordeum vulgare) lines, depicting decreased tocopherol levels and therefore relatively unaltered total tocochromanol levels (Chen et al., 2017). Downstream reactions follow a similar pattern for both tocopherols and tocotrienols, as the catalysis is performed by shared enzymes. Cyclization of MPBQ and MGGBQ results in δ-tocochromanols (δ-tocopherol and δ-tocotrienol, respectively), a reaction which is executed by tocopherol cyclase (TC, VTE1) (Porfirova et al., 2002; Semchuk et al., 2009). However, MPBQ and MGGBQ can take a different route by methyltransferase reactions (MPBQMT, VTE3), resulting in the formation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ) and 2,3-dimethyl-6-geranylgeranyl-1,4-benzoquinone (DMGGBQ) (Cheng et al., 2003). Cyclization of these products by the aforementioned TC results in the formation of γ-tocochromanols. These γ-tocochromanols and δ-tocochromanols can thereafter be methylated by γ-tocopherol methyltransferase (γ-TMT,VTE4) to α-tocochromanols and β-tocochromanols, respectively (Bergmuller et al., 2003). ### Vitamin E Functions in planta Scavenger of Lipid Peroxyl Radicals The most important role of vitamin E in vivo is the termination of a chain reaction of polyunsaturated fatty acid (PUFA) free radicals generated by lipid oxidation. Hence, they play a vital role in scavenging lipid peroxyl radicals during germination and early seedling growth. The detrimental decrease in germination potential of TC mutants (vte1-1) show they are indispensable to preserve the viability of seeds during seed quiescence, which might explain the elevated level of γ-tocopherol in seeds (Sattler et al., 2004). The upstream biosynthesis mutant vte2, which lacks the intermediary DMPBQ, displays difficulties in early seedling development attributable to a decrease in both synthesis and catabolism of lipids as well as an increase in lipid oxidation (Sattler et al., 2004). The few vte2 plants that survive up to the adult stage display no phenotypical differences from wild type which is explained by a predominant need for tocopherols during early development when essential carbon is recruited from lipid catabolism and gluconeogenesis. At later stages, other antioxidants can mitigate the deficiency of tocopherol-mediated ROS scavenging. Hence, tocopherols and its precursors are important to attenuate lipid peroxidation at specific developmental or stress-related periods (Sattler et al., 2006). ### Antioxidant, Photoprotectant, and Stress Signaling The antioxidant function of tocopherols is supported by the ascorbate-glutathione cycle which recycles tocopheroxyl radicals produced during the reaction of tocopherols with lipid peroxyl radicals. Moreover, tocochromanols are, albeit with a lower rate constant than carotenoids, quenchers of singlet oxygen (1O2) (Kaiser et al., 1990). Up to 120 molecules of <sup>1</sup>O<sup>2</sup> can be neutralized by one molecule of α-tocopherol through resonance energy transfer (Fahrenholtz et al., 1974). Related to their scavenging capability, tocochromanols have strong photoprotective properties. When exposing the alga Chlamydomonas to high light, the inhibition of HPP-dioxygenase led to decreased levels of α-tocopherol and concomitantly, to the inactivation of PSII (Trebst et al., 2002). Addition of synthetic, cell-wall permeable, short-chain tocopherol derivatives could partly restore photosynthesis, hence tocopherols are implicated in the maintenance of PSII function, supplemental to the photoprotective function of NPQ (Trebst et al., 2002; Havaux et al., 2005; Kruk et al., 2005). Thus, tocochromanols together with carotenoids and zeaxanthin are the major protectors of PSII vitamins, in dark red, are indicated with a double and a dashed arrow, respectively. Filled yellow boxes indicate the inducers, regulating enzymes surrounded by a yellow square. Tyr, tyrosine; TAT, tyrosine aminotransferase; HPP, p-hydroxyphenylpyruvic acid; HPPD, HPP dioxygenase; PDS1, PHYTOENE DESATURATION1; HGA, homogentisic acid; GGDR, geranylgeranyl diphosphate reductase; HGGT, geranylgeranyl transferase; HPT, homogentisate phytyltransferase; VTE, VITAMIN E PATHWAY gene (1–6); GGPP, geranylgeranyl pyrophosphate; PPP, phytyl pyrophosphate; MGGBQ, 6-geranylgeranyl-benzoquinol; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinone; MPBQMT, MPBQ methyltransferase; DMGGBQ, 2,3-dimethyl-6-geranylgeranyl-1,4-benzoquinone; DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinone; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase; α-,β-,γ-,δ-toc, α-,β-,γ-,δ-tocopherol; α-,β-,γ-,δ-T3, α-,β-,γ-,δ-tocotrienol; SA, salicylic acid; ABA, abscisic acid. against photoinhibition, as they control D1 protein degradation by scavenging singlet oxygen molecules in PSII, and they also protect the whole thylakoid membrane against photooxidative stress, by controlling lipid peroxidation (Trebst et al., 2004). In young leaves of a carotenoid mutant devoid of zeaxanthin, high light stress induced accumulation of tocopherols, conferring tolerance to the mutant, suggesting overlapping functions for these antioxidants (Havaux et al., 2000; Golan et al., 2006). Recently, it was found that an oxidation product, tocopherol quinone, can function as an indicator of oxidative stress, transforming into a signal for programmed cell death upon severe stress. Herewith, the plant protects itself from propagation of stress from the infection point (Li Y. et al., 2008). Moreover, defense-related genes were expressed at higher levels in vte2 plants in response to an increase in peroxidized lipids, suggesting that tocopherol plays a role in gene regulation and modulation of defense responses (Sattler et al., 2006). In this respect, α-tocopherol was found to be important in the mitigation of salt and heavy metal stresses (Jin and Daniell, 2014). In rice, expression of the VTE1 gene was induced by high salt, H2O2, drought and cold, while overexpression led to increased tolerance to salt stress (Ouyang et al., 2011). Conversely, tocopherol deficient Arabidopsis mutants displayed similar phenotypes as wild types under most stress conditions (high light, salinity and drought) applied (Maeda et al., 2006). Hence, in case of tocopherol shortage, other antioxidants can take over its role in stress, yet, vitamin E is an additive value in harsh conditions. ### Membrane Fluidity and Phloem Transport Besides their role as lipophilic antioxidant, tocochromanols also act as important structure-stabilizing agents of membranes (Wang and Quinn, 1999). Their concentration in the chloroplast is most probably tightly regulated as a low concentration of α-tocopherol, comparable with the physiological plastidial concentration, seemed to have an important effect on membrane stability during freezing (Hincha, 2008). On that account tocopherols help, together with other components, to maintain the fluidity and thus the function of photosynthetic membranes. Furthermore, tocopherols have been suggested to play a role in the regulation of photoassimilate export and thus be involved in carbohydrate metabolism, source-sink relationships and growth (Sattler et al., 2003; Hofius et al., 2004). In that respect, a tocopherol cyclase mutant of maize sucrose export defective1 (sxd1) suggested the link between the tocopherol pathway and carbohydrate metabolism as it accumulated carbohydrates in leaves (Russin et al., 1996). The same was observed in StSXD1 RNA interference knockdown lines in potato, but surprisingly not in the vte1 mutant in Arabidopsis, suggesting species-specific differences to tocopherol reduction or a possible additional role of tocopherol in signal transduction (Sattler et al., 2003; Hofius et al., 2004; Li Y. et al., 2008). The biosynthesis mutants vte2 and, to a lesser extent, vte1 revealed inhibition of photoassimilated carbon transport at low temperatures and thus indicated a crucial role of tocopherol in low-temperature adaptation. Cold, non-freezing conditions resulted in a dramatic growth reduction and seed production in these mutants due to structural changes in the phloem parenchyma transfer cells induced by callose deposition and thus leading to reduced photoassimilate export. Lipid peroxidation and photoinhibition were not intensified in vte2, leading to the conclusion that vitamin E function in phloem transport might be more important than its photoprotective role. Apparently the intermediate redoxactive DMPBQ can compensate for the absence of tocopherols as the phenotype of vte1 is not as pronounced as of vte2 (Maeda et al., 2006). ### Vitamin E in Human Health Function and Onset of Deficiency As antioxidants, the different E-vitamers play an important role in neutralizing ROS and inhibiting membrane peroxidation, very much like they do in plants. Due to their amphipathic character, they reside in the membranes, where they perform their peroxyl scavenging function (Brigelius-Flohe, 2009). The main role of these vitamers is to maintain the integrity of long-chain polyunsaturated fatty acids, thereby ensuring their bioactivity (Traber and Atkinson, 2007). Vitamin E deficiency can induce changes in phospholipid composition of membranes, possibly leading to reduced fertility (Infante, 1999). Indeed, vitamin E, together with the micronutrient selenium, has been suggested to serve as a supplement to treat male infertility (Keskes-Ammar et al., 2003). Though tocopherols, predominantly α-tocopherols, are present at higher levels in the human body, significance of tocotrienols should not be neglected (Sen et al., 2006; Colombo, 2010). Indeed, tocotrienols have shown to be effective in inhibiting proliferation of cancers (Aggarwal et al., 2010; Kannappan et al., 2012), albeit that the ability to impede tumorigenesis also has been documented for tocopherols (Li et al., 2011). Vitamin E is also known to have a positive effect on human health by negatively influencing the occurrence of atherosclerosis and cardiovascular diseases (Mathur et al., 2015). Furthermore, vitamin E, α-tocopherol in this case, was shown to delay the development of Alzheimer's disease in patients (Dysken et al., 2014; La Fata et al., 2014). Indeed, vitamin E deficiency aggravates or even induces neurodegenerative disorders (Berman and Brodaty, 2004; Wysota et al., 2017). Hence, vitamin E has been proposed as a therapeutic agent for Alzheimer's disease (Ibrahim et al., 2017). Vitamin E deficiency can impair cognitive functioning, particularly in elderly people (Ortega et al., 2002), which could be explained by aberrant brain energy metabolism, also known to be associated with thiamin deficiency (Sang et al., 2018; Strobbe and Van Der Straeten, 2018) and phospholipid composition (McDougall et al., 2017). ### Global Vitamin E Status Vitamin E deficiency, though not often identified as the causative agent of pathophysiological disorders, is known to be highly prevalent in different populations. Strikingly, a vast majority of the US population is characterized by insufficient intake of dietary α-tocopherol (Maras et al., 2004), the predominant dietary source of vitamin E (Chun et al., 2006). Assessment of vitamin E intake in the French and Italian population, indicated a significant prevalence of suboptimal vitamin E levels (Polito et al., 2005). Interestingly, vitamin E status of the Italian population appeared superior compared to the French, which could be attributed to the typical dietary habits in the Italian culture (see below). More recently, approximately onefourth of the Korean population (in the Seoul metropolitan area) was found to be vitamin E deficient, based on plasma α-tocopherol levels (Kim and Cho, 2015). Furthermore, analysis of blood α-tocopherol levels, confirmed the presence of vitamin E deficiency in many developing countries (Dror and Allen, 2011). ### Sources of Vitamin E Good plant-based sources of dietary (bioactive) vitamin E, in some cases interpreted as supply of α-tocopherol, are fat and oily products such as dried nuts, seeds and almonds (Maras et al., 2004). Tomatoes, avocadoes, spinach, and olives deliver a significant portion of vitamin E (Chun et al., 2006). Though vegetables are generally not a good source of vitamin E (α-tocopherol), soybean and dark leafy greens do exhibit relatively high tocochromanol content. This could explain the rather high vitamin E status of the Italian population (Polito et al., 2005), given the consumption of vitamin E-rich vegetable oil in this region (Huang and Sumpio, 2008). Indeed, the traditional Mediterranean diet has been associated with health benefits, similar to vitamin E, such as reduced incidence of cardiovascular diseases and decreased lipid oxidation (Fito et al., 2007). Starchy, energy-rich staples on the other hand, can be considered rather poor contributors to dietary the vitamin E supply (Table 1). ### Vitamin E Biofortification fpls-09-01862 December 15, 2018 Time: 15:9 # 17 ### Metabolic Engineering Biofortification to enhance vitamin E content in different crops has been successfully deployed over the last decades (Mène-Saffrané and Pellaud, 2017). To understand the rationale behind these strategies, one must first consider the different biological activities of the E-vitamers. As mentioned above, in many cases, α-tocopherol is considered the most potent, bioactive E-vitamer, as confirmed in a rat fetal resorption assay (Bunyan et al., 1961; Mène-Saffrané and Pellaud, 2017). Interestingly, important vitamin E sources such as vegetable oils (soybean, corn, canola and palm) contain a high ratio (up to 10:1) of γ-tocopherol over α-tocopherol (Eitenmiller, 1997). As α-tocopherol was determined to be ten times more bioactive as compared to γ-tocopherol, the idea arose to design metabolic engineering approaches shifting this ratio toward an enhanced relative α-tocopherol content (Shintani and DellaPenna, 1998). However, this objective needs to be justified by assessing the bioavailability as well as storage stability of these vitamers. Indeed, no compelling differences in bioavailability of these E-vitamers were found (Reboul et al., 2008; Reboul, 2017). Unfortunately, α-tocopherol appears less stable in storage, as it reacts faster with peroxy radicals, confirmed by the higher instability of α-tocopherol compared to γ-tocopherol in storage of camelina (Camelina sativa) oil (Abramovic et al., 2007). Although this issue should not be neglected, the higher bioactivity of the α-tocopherol vitamer could outweigh this disadvantage. Introduction of a γ-tocopherol methyltransferase (γ-TMT) (Figure 5) (Tewari et al., 2017), catalyzing the addition of the required methyl group to form α-tocopherol from γ-tocopherol (Figures 4, 5), was therefore conducted. This strategy was proven successful in Arabidopsis, where the α/γ-tocopherol ratio was completely reversed in favor of α-tocopherol accumulation in seeds overexpressing the γ-TMT gene (Shintani and DellaPenna, 1998; Mène-Saffrané and Pellaud, 2017). This strategy has been implemented in several crops, including corn (Zhang L. et al., 2013), soybean (Glycine max) (Arun et al., 2014) and lettuce (Lactuca sativa L.) (Cho et al., 2005). Theoretically, the biological activity of the crop vitamin E pool can be increased up to 10-fold by this strategy (Mène-Saffrané and Pellaud, 2017). In rice endosperm, ectopic γ-TMT expression yielded no significant change in α-tocopherol content, explained by low γ-tocopherol levels, yet significantly altered tocotrienol levels, in favor of α-tocotrienol (Zhang G.Y. et al., 2013). Interestingly, implementation of this metabolic engineering approach, yielding higher α-tocopherol content in alfalfa leaves (Medicago sativa), coincided with a delayed leaf senescence phenotype as well as enhanced tolerance to osmotic stress (Jiang et al., 2016). As this strategy does not greatly influence accumulation of the absolute tocochromanol content, applicability is confined to crops accumulating higher levels of E-vitamers with lowered bioactivity, such as γ-tocopherols and δ-tocopherols. Furthermore, generalization of E-vitamers into absolute values of 'bioactivity' could prove to be difficult. Indeed, different vitamers could exhibit different potencies in a whole range of biological functions, but without a single vitamer being omnipotent. This is indicated by the observed higher ability of γ-tocopherol to reduce 8-isoprostane [oxidative stress marker (Elfsmark et al., 2018)] (Jiang et al., 2002; Jiang and Ames, 2003). Assigning a universal (vitamin E) bioactivity to a specific vitamer could miss identifying its full biological potential. Moreover, the typical accumulation of γ-tocopherols witnessed in seeds (Sattler et al., 2004; Gilliland et al., 2006) (and therefore contributing to the vitamin E content of oils), might hint at its physiological importance in planta. Fortunately, no aberrant growth and fertility have been reported in the γ-TMT-engineered biofortified crops, indicating that the altered tocopherol ratio has marginal effects on plant growth and development (Mène-Saffrané and Pellaud, 2017). Besides redirection of tocopherol homeostasis toward a more satisfactory vitamer composition, increase of (absolute) vitamer content has been tackled in metabolic engineering approaches (Cahoon et al., 2003). Engineering the HGGT gene, catalyzing the committed step in tocotrienol biosynthesis (Figure 5), resulted in an increase in total tocochromanol content of maize kernels and up to 18-fold enhancement in tocotrienol accumulation (Dolde and Wang, 2011). Furthermore, engineering HPPD, a key enzyme in the biosynthesis of the tocochromanol precursor HGA (Figure 5), generated a massive accumulation of tocotrienols, provided that prephenate dehydrogenase (shikimate pathway) was also engineered to ensure sufficient flux toward tyrosine (Rippert et al., 2004). Building further on this approach, high tocochromanol accumulating soybean was created via additional introduction of HPT and GGDR (see Figure 5) (Karunanandaa et al., 2005). However, biofortification approaches should not neglect tocochromanol stability, as vitamin E levels were shown to halve in freeze-dried fortified apple upon 6 months storage (Cortes et al., 2009). Further details on the different strategies employed in biofortification of crops toward higher vitamin E content have been elaborated by Mène-Saffrané and Pellaud (2017). ### Breeding From the perspective of plant breeders, an interesting amount of variation in vitamin E content has been observed in different agronomical important crops (Mène-Saffrané and Pellaud, 2017). In rice, total kernel vitamin E content was found to vary up to threefold in different Malaysia-grown varieties (Shammugasamy et al., 2015). Similarly, a study in canola, which is important for oil production and therefore tocochromanol delivery, identified VTE3 and PDS as important determinants of tocopherol content, based on screening of 229 accessions (Fritsche et al., 2012). Moreover, a measured variation of almost sixfold in maize kernel α-tocopherol content enabled conducting a GWAS wherein a HGGT gene, a prephenate dehydratase paralog [participating in tyrosine biosynthesis (El-Azaz et al., 2016)] and a tocopherol cyclase were recognized to contribute to tocotrienol content (Lipka et al., 2013). The same study further confirmed the link between γ-TMT alleles and α-tocopherol content. Interestingly, more recent GWAS in maize revealed many significant QTL loci, attributed to genes harboring novel activities as well as participating outside the tocopherol pathway (Diepenbrock et al., 2017; Wang et al., 2018). In conclusion, this is a nice example of GWAS and assignment of candidate genes to the identified QTLs to pinpoint potential factors for novel metabolic engineering approaches. ### Tocochromanols: Major Problems and Future Perspectives The case of tocochromanols, comprising tocopherols and tocotrienols, is a good example on how simplifying these distinct groups of molecules to their collective term 'vitamin E' can be misleading. As previously mentioned, the bioactivity of E-vitamers is diverse. However, bioactivity alters depending on which tocochromanol-related process is utilized to assess it. Moreover, there is no one-to-one relationship between a certain vitamer and a given function. One could therefore argue that grouping tocochromanols into one group of 'vitamin E' is incorrect. This notion becomes more important given the existence of different metabolic engineering approaches aimed at altering E-vitamer ratios (e.g., increasing α-tocopherol/γ-tocopherol ratio) while keeping total tocochromanol levels intact (γ-TMT-engineering). Similarly, bioavailability as well as (storage) stability should not be neglected. Moreover, whether engineering approaches are based on altering tocochromanol ratio (e.g., via γ-TMT-engineering) or enhancing total tocochromanol content (e.g., HGGTengineering), the impact on plant growth and development should be closely monitored. Finally, seeds, being an important target for metabolic engineering approaches, often depict a typical tocochromanol signature (Sattler et al., 2004), related to their function therein, which could be disrupted upon engineering approaches. Future research should therefore further unravel the in planta role of the different vitamin E entities. Similarly, the pathophysiological significance of the different vitamers in humans should be thoroughly examined. ### INTERTWINING OF VITAMIN METABOLISM AND ITS SIGNIFICANCE IN MULTI-BIOFORTIFICATION A simultaneous increase of several micronutrients in a particular crop/tissue, referred to as multi-biofortification, is a powerful means to tackle MNM. This strategy aims at obtaining adequate levels of multiple micronutrients in a single staple crop, which is massively consumed by the local population in need. Such endeavor might encounter synergistic but also potentially detrimental effects, due to micronutrient interactions. Taking the example of the antioxidant ascorbate, protection of components sensitive to oxidative damage (e.g., carotenoids) is expected, thereby contributing to their accumulation as well as stability upon storage, an advantage which could also be expected from the combination with vitamin E. Furthermore, the ascorbate-glutathione pathway is needed in the 'detoxification' of tocopheroxyl radicals in vitamin E salvage (Szarka et al., 2012). In addition, ascorbate is known to ameliorate iron uptake in humans (Iqbal et al., 2004). Consequently, ascorbate biofortified crops could also aid in combatting iron deficiency indirectly. Similarly, provitamin A and vitamin E biofortification have shown to be positively affect one another (Che et al., 2016; Muzhingi et al., 2017). In the example of biofortified sorghum, the raised level of vitamin E, obtained by genetic engineering, enhanced provitamin A stability (Che et al., 2016). Interestingly, a synergistic interrelationship between ascorbate and vitamin B9 (folates) has been proposed, justified by their coextensive increase during germination (Liu et al., 2017). This study also proposes that folates (vitamin B9) biosynthesis counteracts vitamin E biosynthesis by its competition for the precursor GTP. Competition for precursors could prove to have a substantial influence on vitamin metabolism, considering the fact that vitamin E biosynthesis requires precursors from shikimate and MEP pathways, which are also required in the folate and provitamin A pathways, respectively. Conversely, folates are proposed to aid in maintaining high ascorbate content, as they contribute in supplying NADPH to the cell, which could support adequate ascorbate salvage (Gorelova et al., 2017; Liu et al., 2017). Moreover, DXS activity, which has been enhanced in different metabolic engineering approaches aimed at augmenting plant provitamin A content, requires active B1 vitamer cofactor (thiamin pyrophosphate) for its functioning (White et al., 2016) and is also required in the biosynthesis of tocochromanols. This nicely illustrates how different vitamins are part of a potentially strong network of interactions in plant as well as in human metabolism. This aspect certainly deserves proper consideration upon evaluation of novel biofortification strategies (Strobbe and Van Der Straeten, 2018). Furthermore, certain environmental influences could alter the accumulation of multiple vitamins, illustrated by the light-dependent accumulation of both provitamin A and tocochromanols (Cruz et al., 2018; Gramegna et al., 2018). This aspect can therefore be considered upon setting light conditions in vertical farming projects (Bantis et al., 2018). Last but not least, biofortification could have the beneficial 'side-effect' of enhancing tolerance to abiotic stresses, as reported in metabolic engineering approaches enhancing plant ascorbate content (Macknight et al., 2017). This is particularly important given the increased exposure to abiotic stresses, but also to biotic stresses crops will have to face as a result of climate change (Cheeseman, 2016). ## CONCLUSION fpls-09-01862 December 15, 2018 Time: 15:9 # 19 Vitamin biofortification of food crops holds the potential to alleviate the global burden of vitamin deficiencies (Blancquaert et al., 2017; Garcia-Casal et al., 2017; Jiang L. et al., 2017; Martin and Li, 2017; Van Der Straeten et al., 2017; Garg et al., 2018). In doing so, staple crops will play a predominant role, as they hold the impressive capability to deliver cheap calories to populations in need and have the potential to be nutritionally enhanced via metabolic engineering or breeding approaches. Both conventional breeding and metabolic engineering should coexist in the battle against vitamin deficiencies, thereby reciprocally strengthening their potential. Molecular breeding techniques such as GWAS promise to facilitate enhancement of crop vitamin content whilst uncovering potential new determinants in vitamin accumulation in the particular crop/tissue, subsequently applicable in new engineering approaches. In some cases, downregulation of genes impeding vitamin accumulation is advised (see provitamin A biofortification). Here, metabolic engineering strategies utilizing genome-editing techniques such as the CRISPR/Cas system are promising, especially considering they might suffer less from regulatory issues blocking their commercialization (Potrykus, 2017), in cases where no transgenes are introduced. However, this technology still faces crop-specific limitations toward the maximal vitamin enhancement possible. Therefore, a combination with metabolic engineering strategies employing transgenes, is advisable, in which CRISPR/Cas technology could still be utilized to allow specific T-DNA insertion the genome position of interest. When using a biofortification approach, several aspects should be considered, including bioavailability, bioactivity, stability and impact on crop yield and/or physiology. Bioavailability, bioactivity and stability can be addressed by ### REFERENCES examination of these properties on the specific biofortified crop, and targeted by specific strategies to confer these properties to the crop product [e.g., engineering toward more stable folates in rice (Blancquaert et al., 2015), or engineering toward more potent α-tocopherol (Shintani and DellaPenna, 1998)]. Assessment of biofortification interventions influencing plant physiology (and thereby yield) requires indepth analysis and knowledge of micronutrient metabolism as well as post hoc examination of plant physiology in field conditions. Given their potential to provide sufficient micronutrients, (multi-)biofortified crops are a crucial piece of the puzzle in eradicating micronutrient deficiencies on a global scale. Moreover, biofortified crops are already contributing to sustainable food security in a time of increasing global demographic pressure and climate change. Last but not least, they hold great potential to contribute even more to maintaining a healthy world population into the future, provided that novel approaches to biofortification are embraced. ### AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. ### FUNDING SS was indebted to the Agency for Innovation by Science and Technology in Flanders (IWT) for a predoctoral fellowship. DV acknowledges support from Ghent University (Bijzonder Onderzoeksfonds, BOF2009/G0A/004, BOF2018/GOA/042), and the Research Foundation—Flanders (FWO, project 3G012609). a plant ascorbic acid biosynthetic enzyme. J. Biol. Chem. 281, 15662–15670. doi: 10.1074/jbc.M601409200 enhanced seed tocopherol. Metab. Eng. 7, 384–400. doi: 10.1016/j.ymben.2005. 05.005 fruit metabolic traits. Plant Physiol. 165, 1120–1132. doi: 10.1104/pp.114.24 1521 dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci. 164, 1111–1117. doi: 10.1016/S0168-9452(03)00122-5 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Strobbe, De Lepeleire and Van Der Straeten. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Natural Variation in Physiological Responses of Tunisian Hedysarum carnosum Under Iron Deficiency Heithem Ben Abdallah<sup>1</sup> , Hans Jörg Mai<sup>1</sup> , Tarek Slatni<sup>2</sup> , Claudia Fink-Straube<sup>3</sup> , Chedly Abdelly<sup>2</sup> and Petra Bauer1,4 \* 1 Institute of Botany, Heinrich Heine University Düsseldorf, Düsseldorf, Germany, <sup>2</sup> Laboratory of Extremophile Plant, Center of Biotechnology of Borj Cedria, Hammam-Lif, Tunisia, <sup>3</sup> INM-Leibniz Institute for New Materials, Saarbrücken, Germany, <sup>4</sup> Cluster of Excellence on Plant Sciences, Heinrich Heine University, Düsseldorf, Germany Iron (Fe) is an essential element for plant growth and development. The cultivation of leguminous plants has generated strong interest because of their growth even on poor soils. Calcareous and saline soils with poor mineral availability are wide-spread in Tunisia. In an attempt to select better forage crops adapted to Tunisian soils, we characterized Fe deficiency responses of three different isolates of Hedysarum carnosum, an endemic Tunisian extremophile species growing in native stands in salt and calcareous soil conditions. H. carnosum is a non-model crop. The three isolates, named according to their habitats Karkar, Thelja, and Douiret, differed in the expression of Fe deficiency symptoms like morphology, leaf chlorosis with compromised leaf chlorophyll content and photosynthetic capacity and leaf metal contents. Across these parameters Thelja was found to be tolerant, while Karkar and Douiret were susceptible to Fe deficiency stress. The three physiological and molecular indicators of the iron deficiency response in roots, Fe reductase activity, growth medium acidification and induction of the IRON-REGULATED TRANSPORTER1 homolog, indicated that all lines responded to −Fe, however, varied in the strength of the different responses. We conclude that the individual lines have distinct adaptation capacities to react to iron deficiency, presumably involving mechanisms of whole-plant iron homeostasis and internal metal distribution. The Fe deficiency tolerance of Thelja might be linked with adaptation to its natural habitat on calcareous soil. Keywords: legume, natural diversity, iron deficiency, chlorophyll, acidification, Fe reductase activity, IRT1 ### INTRODUCTION Iron (Fe) is an essential micronutrient with numerous cellular functions, e.g., in photosynthesis, respiration, DNA synthesis, and N<sup>2</sup> fixation. Plants are frequently challenged by Fe deficiency, especially on alkaline and calcareous soils due to poor Fe solubility under these conditions. In Tunisia, the exploration of such kinds of natural habitats and saline environments revealed that they are colonized by a native leguminous vegetation which might have specific adaptations to both, salinity and nutrient deficiencies, especially Fe (Ben Abdallah et al., 2017). Leguminous plants take up reduced Fe using mainly the so-called Strategy Edited by: Huixia Shou, Zhejiang University, China #### Reviewed by: Ping Lan, Institute of Soil Science (CAS), China Marta Dell'Orto, Università degli Studi di Milano, Italy > \Correspondence: Petra Bauer [email protected]; #### Specialty section: [email protected] This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 08 December 2017 Accepted: 30 August 2018 Published: 02 October 2018 #### Citation: Ben Abdallah H, Mai HJ, Slatni T, Fink-Straube C, Abdelly C and Bauer P (2018) Natural Variation in Physiological Responses of Tunisian Hedysarum carnosum Under Iron Deficiency. Front. Plant Sci. 9:1383. doi: 10.3389/fpls.2018.01383 I. The main feature of Strategy I plants, e.g., in Arabidopsis thaliana and leguminous plants, is that they acidify the soil via proton extrusion through an ATPase, reduce ferric to ferrous Fe by a ferric chelate reductase and take up the divalent Fe via divalent metal IRON-REGULATED TRANSPORTER1 (Brumbarova et al., 2015), being a member of the ancient ZIP ( = ZRT/IRT1) protein family (Eng et al., 1998). IRT1 homologs were found Fe-regulated in roots of multiple legumes like Pisum sativum (Cohen et al., 2004), Medicago truncatula (Lopez-Millan et al., 2004), Arachis hypogaea (Ding et al., 2010), Glycine max (Brear et al., 2013), and Vigna radiata (Muneer et al., 2014). Natural variation studies make use of the existing natural allelic diversity in plant populations as a source to pinpoint the adaptive alleles for relevant traits. Natural variation was successfully applied in model plants to identify causal alleles by genome-wide association studies for such different traits as environmental adaptation in Arabidopsis (Li et al., 2010), or nutritional quality and agronomic traits in maize (Diepenbrock et al., 2017) and rice (Si et al., 2016). The model legumes M. truncatula and G. max are particularly suited for natural biodiversity studies (Gentzbittel et al., 2015). Prerequisites for association studies are genome sequence variation reflected by a broad collection of ecotypes demonstrating phenotypic diversity for given traits. However, alternative procedures are available for studying natural diversity of small population collections in the absence of large genome sequence data, e.g., by making use of recombinant or near-isogenic inbred lines suitable for mapping and gene identification (Yan et al., 2017), by isolating candidate genomic regions and genes based on comparative genomics (Friesen et al., 2010, 2014; Turner et al., 2010) and transcriptomics or proteomics of genetically divergent lines (Voelckel et al., 2017). In Tunisia, a large area is arid to semi-arid with calcareous and alkaline-saline soil conditions, where bioavailability of Fe, Mg, and other minerals is low, posing a major problem for crop yield (Rabhi et al., 2007). Such areas are often used for forage crop and cattle production. To improve agricultural land usage and provide better perspectives to farmers, there is a need to select tolerant crops adapted to such poor soils. Perennial Hedysarum carnosum was proposed as a prospective well-palatable pasture crop being a naturally adapted halophyte to the Mediterranean basin with good potential for rehabilitation strategies (Le Houerou, 1996). The Hedysarum (sweetvetch) genus belongs to the Fabaceae plant family, which contains many of the very important crops. Species of this genus have arisen in a non-monophyletic manner, as recently established based on multiple sequence alignments and phylogenetic tree constructions of 58 accessions accounted to this genus using nuclear and plastid gene sequences (Liu et al., 2017). H. carnosum (also known as Sulla carnosa) that is subject of this study is most related to Hedysarum coronarium (also known as Sulla coronaria) (Liu et al., 2017). H. coronarium is wide-spread around the Mediterranean basin. In contrast, H. carnosum is endemic in Tunisia where it grows in different climates, ranging from semi-arid (Karkar) to arid regions (Thelja and Douiret). This species prefers slightly acid to alkaline soils (pH 5.5–8.5), sandy loams and clays, and good growth is achieved on alkaline soils. As an extremophile, H. carnosum also grows on Tunisian saline sodic soils, represented especially by Chotts and Sebkhas (Dallali et al., 2012). This species plays important roles in animal feed due to its high protein contents and tannins (Aissa et al., 2016). H. carnosum is a non-model crop and molecular investigation is difficult because of the lack of molecular data and gene sequences. H. carnosum responds to salt, potassium and magnesium deficiencies and low Fe availability (Farhat et al., 2016; Elkhouni et al., 2017; Hafsi et al., 2017). However, in all these studies only a single H. carnosum line was studied, rendering it difficult to judge the natural adaptation potential within the species. Moreover, these studies primarily focused on photosynthetic parameters in leaves. The objective of this work was to investigate the effect of Fe deficiency at the physiological level in H. carnosum and to compare the responses in three different isolates collected from different natural sites. Our data show that the Thelja isolate is the most tolerant to Fe deficiency stress and therefore will be the best choice for use in future genetic and RNAseq studies to identify the natural basis for calcareous soil-induced Fe deficiency. Thelja will also represent the most promising ecotype for agricultural purposes. ### MATERIALS AND METHODS ### Plant Material and Growth Condition Three isolates of H. carnosum were acquired by collecting seeds from Karkar, Thelja, and Douiret in Tunisia (see Supplementary Figure 1* for species characteristics and geographic repartition of collected isolates). About 300 seeds per isolate were collected from an average of 10 plants distributed in a diameter of 100 m. Seeds of the three isolates were germinated and grown in separate green houses. Due to allogamy plants were multiplied by crossfertilization of plants within each isolate. The F3 generation was used for analyses. Seeds were mechanically scarified by rubbing in between fine grit sand paper sheets. The seeds were sterilized in 10% sodium hypochlorite for 8 min and then abundantly rinsed with distilled water. After a 10 min imbibition phase, they were germinated for 4 days at 20◦C in Petri dishes on constantly moistened filter paper. Four day-old seedlings were transferred to a half strength aerated liquid nutrient solution for 2 days. Similarly sized seedlings were then selected and cultured in groups of 4 or 10 plants in 1 or 10 L of full strength aerated nutrient solution [1.5 mM Ca(NO3)2, 1.25 mM KNO3, 0.75 mM MgSO4, 0.5 mM KH2PO<sup>4</sup> and 10 µM H3BO3, 1 µM MnSO4, 0.5 µM ZnSO4, 3 µM MOO4Na2, 0.5 µM CuSO4, and 50 µM Fe-EDTA]. At day 6, the following treatments were conducted for the amount of time indicated in the text and figure legends: +Fe, control (Fe-sufficient medium with 50 µM Fe) and −Fe, Fe deficiency (medium without Fe). The pH was adjusted to 6.0 with NaOH for both, the +Fe (control) and −Fe (iron deficiency) treatments. Aerated hydroponic cultures were maintained in a growth chamber with a day/night regime of 16/8 h light-dark-cycle, a 24/18◦C temperature cycle and a constant relative humidity of 70%. The FIGURE 1 \| Effect of Fe deficiency stress on Karkar, Thelja, and Douiret lines. (A) Root dry weight (RDW), n = 3; (B) shoot dry weight (SDW), n = 3; (C) main root length, n = 4–5; (D) total chlorophyll (Chl) content, n = 3; (E,F) photon yield of PSII calculated by Fv/Fm and Fv/F0, n = 4; (G) chlorosis phenotype symptoms of young leaves calculated according to the indicated scale from 1, green leaves up to 5,white-yellow leaves, n = 4. H. carnosum plants were exposed for 10 days (A–F) or up to 10 days (G) to Fe-sufficient (+Fe) and Fe-deficient (–Fe) hydroponic growth conditions. Data are means ± SD; means with the same letter are not significantly different with P ≤ 0.05 according to ANOVA and Tukey's HSD test. solution was renewed every 4 days. The standard experiment was conducted using ten-day Fe sufficiency and deficiency treatments. ## Morphological Root and Shoot Phenotypes Roots and shoots were harvested, dried in an oven at 70◦C for 48 h and the dry weights determined per plant. The main root lengths were measured. The degree of leaf chlorosis was assessed in the youngest expanded leaves. The leaf chlorosis scale was determined as previously described (Schuler et al., 2012), ranging from 1 = green, 2 = light green, partially yellow, 3 = yellow-green, 4 = yellow to 5 = white-yellow, as shown in Figure 1G. ### Chlorophyll Measurements Total chlorophyll was extracted from fresh leaves in 80% acetone and assayed photometrically at 645 nm and 663 nm. The OD values were used to calculate the total chlorophyll content in mg/g fresh weight of the leaves as published (Arnon, 1949). ### Pulse Amplitude Measurements (PAM) Pulse Amplitude Measurements (PAM) was determined with the FluorCam FC 800-C machine (Photon Systems InstrumentsTM). Plants were adapted to darkness for about 15 min. Then single leaves were measured for F<sup>0</sup> (minimal fluorescence) up to Fm (maximal fluorescence). To analyze photosystem II activity, Fv/Fm values and Fv/F<sup>0</sup> values were calculated (Murchie and Lawson, 2013). F<sup>0</sup> and Fm represent the minimum and maximum values of chlorophyll fluorescence, while F<sup>V</sup> is the variable fluorescence. ### Acidification of the Growth Medium The acidification capacity was determined after placing plants into 1 L nutrient solution (at pH 6.2), respectively, and measuring the pH of the nutrient solution in the subsequent days as indicated in the text and figure legend. ### Measurements of Root Fe Reductase Activity Intact root systems were washed with 100 mM Ca(NO3)<sup>2</sup> solution and submerged in the Fe reductase assay solution containing 0.1 mM Fe3+-NaEDTA and 0.3 mM ferrozine at pH 5.0 for 1 h in the dark. Then the absorbance was determined at 562 nm. The concentration of the Fe2+-ferrozine complex was calculated using the molar extinction coefficient of 28.6 mM−<sup>1</sup> cm−<sup>1</sup> . The amount of Fe2<sup>+</sup> was normalized to the root weight in the assay and Fe reductase activity was calculated. ## Mineral Element Analysis To determine the metal ion content the youngest expanded leaves of H. carnosum were harvested and dried over 72 h at 70◦C. After drying, the harvested leaves were finely powdered with an achat mortar and pestle. Metal contents (Zn, Fe, Cu) were determined using inductively-coupled plasma optical emission spectrometry (ICP-OES) at the Leibniz Institute for New Materials (INM, Saarbrücken). FIGURE 2 \| Metal contents of Karkar, Thelja, and Douiret. (A) Fe; (B) Zn; (C) Cu contents of the youngest expanded leaves. H. carnosum plants were exposed for 10 days to Fe-sufficient (+Fe) and Fe-deficient (–Fe) hydroponic growth conditions. Data are means ± SD; n = 4; means with the same letter are not significantly different with P ≤ 0.05 according to ANOVA and Tukey's HSD test. ### Obtention of H. carnosum cDNA Sequences and Multiple Sequence Alignment of Amino Acid Sequences Obtention of H. carnosum cDNA sequences is outlined in Supplementary Figure 2A. A. thaliana, M. truncatula, G. max, Lotus japonicus sequences of IRT1 and β-ACTIN (ACT) were aligned. Conserved regions near the start and stop codons were identified and primers matching 100% the M. truncatula sequences were designed (Mt primers, Supplementary Figure 3). With these Mt primers 1 µL of template root cDNA of H. carnosum was used to amplify the IRT1 and ACT internal coding sequences in a standard PCR. PCR amplicon bands were purified from agarose gels according to standard procedures and sequenced. Next, TAIL-PCR (Supplementary Figure 2B) was used to identify the unknown upstream 5<sup>0</sup> and downstream 3<sup>0</sup> cDNA sequences adjacent to the determined HcIRT1 and HcACT partial sequences. We used three nested specific primers (S1–S3) that aligned near the edge of the known cDNA (Supplementary Figure 3). For extension in the opposite direction AD (arbitrary degenerate) primers were used. The AD primers were 64x–256x degenerate and designed to be relatively short (15–16 nt) with a low melting temperature (ca. 40–50◦C) (Supplementary Figure 3). Three consecutive TAIL-PCR reactions were conducted as described (Liu et al., 1995). The third step PCR products were sequenced and HcIRT1 and HcACT sequences assembled and provided to GenBank (accession numbers MH879027 and MH879028). Multiple sequence alignment and construction of neighborjoining trees using amino acid sequences was performed using the Clustal Omega tool at https://www.ebi.ac.uk. ### RNA Isolation and Quantitative Real-Time PCR Gene expression was analyzed using three biological replicates. RNA isolation and reverse transcription-quantitative PCR were carried out as described previously (Ben Abdallah and Bauer, 2016). Briefly, total RNA prepared from 100 mg H. carnosum root tissue was used for cDNA synthesis using an oligo-dT primer. qPCR was conducted using the SYBR Green detection method. RT-qPCR primers were used for qPCR. The absolute quantity of initial transcripts was determined for the genes IRT1 and ACT by standard curve analysis using mass standards prepared from H. carnosum cDNA PCR products amplified with Mt primers. Absolute expression data of IRT1 was obtained after normalization to the internal control ACT gene. Each biological cDNA sample was tested in two technical qPCR replicates. ### Statistical Analysis Morphological, physiological, and molecular data were obtained in at least three biological replicates, as detailed in the figure legends. Data of biological replicates were used to calculate mean values and standard deviations. Statistical significance was determined by applying t-tests (for two sample comparisons) and One-way ANOVA followed by Tukey's HSD test (for more than two sample comparisons) designated as "ANOVA and Tukey's HSD test" in the figure legends. ## RESULTS ### Morphological and Physiological Shoot Responses to Fe Deficiency Seeds from H. carnosum plants were collected in three different locations in Tunisia characterized by semi-arid, arid and Saharan conditions, named Karkar, Thelja, and Douiret, with salinesodic, calcareous and sandy soil characteristics (Supplementary Figure 1). After seeds were multiplied for three generations, morphological and physiological experiments were carried out. We were interested in obtaining an ecotype with high tolerance to prolonged Fe deficiency growth conditions, a trait expected to be beneficial upon growth on calcareous soil. We therefore hypothesized that the three isolates might show different adaptation and respond differently to Fe deficiency conditions. Plant seedlings were grown in controlled hydroponic conditions and exposed to sufficient iron (+Fe) or deficient iron supply (−Fe) for 10 days. At first, we compared the tolerance/sensitivity of the lines to −Fe by measuring different growth parameters. The three isolates did not behave any different from each other in terms of root biomass production under + and −Fe (Figure 1A). Also in terms of shoot biomass production, the ecotypes were very similar (Figure 1B). Only one comparison resulted in a significant difference in biomass, which was the fourfold higher shoot dry weight of Douiret versus Karkar at −Fe (Figure 1B). However, none of the lines showed lower root or shoot biomass when grown at – compared to +Fe (Figures 1A,B). Karkar displayed a shorter main root compared to Douiret at +Fe. When comparing the main root length at −Fe versus +Fe, there was a significant decrease only in the case of Douiret but not Karkar and Thelja (Figure 1C). Fe is required in high amounts during plant growth in the leaves to sustain photosynthesis and for chlorophyll synthesis. Fe n = 4. H. carnosum plants were exposed for 10 days (A) or up to 10 days (B) to Fe-sufficient (+Fe) and Fe-deficient (–Fe) hydroponic growth conditions. Data are means ± SD; means with the same letter are not significantly different with P ≤ 0.05 according to ANOVA and Tukey's HSD test, means with <sup>∗</sup> label in B show a significant difference of + versus –Fe with P ≤ 0.05 according to a t-test. can also be stored in chloroplasts in the form of ferritin. Lack of Fe results in the typical leaf chlorosis symptoms especially in the expanding leaves. Leaf chlorosis is caused by low chlorophyll contents under Fe deficiency. Karkar and Douiret had higher total chlorophyll contents at + than at −Fe (Figure 1D). Thelja, on the other hand, displayed no significant difference at + versus −Fe (Figure 1D). No significant differences were detectable between the lines at either + or −Fe (Figure 1D). PAM measurements based on chlorophyll fluorescence are an indicator for the photosynthetic performance under stress conditions. Low Fv/Fm and Fv/F<sup>0</sup> ratios are indicative of stress affecting negatively the photosystem activity. We found that Karkar had lower Fv/Fm and Fv/F<sup>0</sup> ratios at – versus +Fe, while no significant differences were found in Thelja and Douiret (Figures 1E,F). When comparing the lines with each other, Karkar had a lower Fv/Fm ratio than Thelja and Douiret and Thelja had a higher Fv/F<sup>0</sup> ratio than Karkar and Douiret (Figures 1E,F). We were also interested in comparing the development of the leaf chlorosis during the 10 days of exposure to −Fe. Leaf chlorosis started 2 days earlier in Douiret than in Karkar and Thelja, but after 10 days the chlorosis had reached similar levels, as determined above from the chlorophyll measurements (Figures 1D,G). Next, we investigated whether the observed leaf chlorosis and impact on photosynthesis could be related to the amount of Fe taken up. We determined metal contents in the expanding leaves, as these are the organs where Fe deficiency symptoms are noted. Roots were not used since under hydroponic growth Fe is available in the Form of Fe Na EDTA, resulting in an accumulation and high Fe content, e.g., in the apoplasts of roots. In addition to Fe we measured Zn and Cu contents. Arabidopsis IRT1 can take up Zn but not Cu (Vert et al., 2002) and MtZIP6 can also transport Zn (Lopez-Millan et al., 2004). Karkar and Douiret had lower Fe contents upon −Fe than under +Fe, but not Thelja, which had comparable levels under both conditions (Figure 2A). Thelja also had higher Fe contents upon −Fe compared to Karkar and Douiret (Figure 2A). The Zn content was decreased at – versus +Fe growth conditions only in Douiret (Figure 2B). However, Thelja had a higher Zn content than Karkar and Douiret at – but not +Fe (Figure 2B). The Cu content was decreased at – versus +Fe in Karkar and Douiret, but again not in Thelja (Figure 2C). When comparing the lines with each other, a significant difference of Cu was only found in the comparison of Thelja versus Karkar at −Fe (Figure 2C). To conclude from this physiological and growth analysis of the three lines exposed to + and −Fe, we summarized the selected legumes and Arabidopsis. The multiple amino acid sequence alignment was produced with H. carnosum IRT1 and all identified ZIP protein sequences from A. thaliana, M. truncatula, G. max, and L. japonicus. The box indicates the closest relatives of AtIRT1. The star indicates HcIRT1. comparative outcomes for the parameters measured at + versus −Fe for each line and designated a significant decrease at – versus +Fe as "sensitive" and no decrease as "tolerant" behavior (Figure 3). Karkar received four sensitivity and five tolerance labels, Douiret six sensitivity and three tolerance labels, and Thelja nine tolerance labels. Root and shoot biomass were not identified as parameters that could be used to discriminate the behavior of the lines at + and −Fe, while leaf chlorosis, photosystem activity and metal contents were well suited to do so. Taken together, it can be deduced that Thelja shows tolerance to −Fe in contrast to the other two lines. ### Physiological and Molecular Root Responses to Fe Deficiency Roots of strategy I plants show typical Fe deficiency symptoms like enhanced soil acidification, Fe reduction and increased IRT1 gene expression. Quantification of these responses is used to judge the degree of tolerance to Fe deficiency (Brumbarova et al., 2015). Therefore, we tested next for potential differences in the level of Fe deficiency responses in the root. None of the plants subjected to Fe deficiency showed a significant increase in root Fe reductase activity (Figure 4A). In a time-course experiment we found that the growth medium was acidified significantly starting as early 2 days after exchange to Fe deficiency and continued until 10 days in all three lines (Figure 4B). H. carnosum gene sequences were not deposited in the database. To conduct gene expression analysis by the RT-qPCR method, we selected IRT1 as a target gene to reflect molecular Fe uptake regulation. Gene expression of additional genes were not investigated here as it is more useful to conduct RNAseq studies in the future. First, we identified homologs of IRT1 and of the reference gene β-ACTIN (ACT) from H. carnosum using PCR and TAIL-PCR by exploiting sequence similarities among leguminous plant IRT1 sequences and available microarray-based gene expression data for M. truncatula (see section "Materials and Methods"; outline in Supplementary Figure 2). Since we found no differences in the amino acid sequences of IRT1 between the three H. carnosum lines, we compared the sequence to ZIP sequences from Arabidopsis and other legumes. The full-length HcIRT1 amino acid sequence was found most related to MtZIP6 in a neighbor-joining tree derived from a multiple sequence alignment of the entire families of A. thaliana, M. truncatula, G. max, and L. japonicus ZIP protein sequences (Figure 5). MtZIP6 was the only M. truncatula ZIP protein with high sequence similarity to HcIRT1 and AtIRT1 (Figure 5). MtZIP6 is up-regulated by −Fe in roots (He et al., 2009; Benedito et al., 2010) and it was characterized as Fe transporter (Lopez-Millan et al., 2004). All other M. truncatula ZIP proteins group along with other branches of A. thaliana ZIP proteins (Figure 5), indicating that these other MtZIP proteins have different functions in metal homeostasis. Interestingly, this analysis also shows that A. thaliana has a high expansion of four IRT1-like proteins (IRT1, IRT2, ZIP8, ZIP10). In this same branch of IRT1-like sequences, there are two G. max, two L. japonicus and only one M. truncatula ZIP sequence (highlighted by a red box in Figure 5). Thus, legumes have fewer IRT1-like proteins than Arabidopsis. One possible explanation could be the different genome duplication histories during evolution. HcIRT1 and MtZIP6 share two important functional sequence features in the predicted variable cytoplasmic loop region with AtIRT1, namely two conserved lysine positions used for ubiquitination in metal-directed IRT1 turnover (Kerkeb et al., 2008) and the histidine-rich stretch for metal-binding relevant for metal import by ZIPs into the cell (Zhang et al., 2017; Figure 6A). Hence, the sequence analysis convincingly suggests that HcIRT1 encodes a functional IRT1 homolog. HcIRT1 gene expression was found significantly induced by −Fe in Karkar, Thelja, and Douiret (Figure 6B). Thelja displayed a higher base level of HcIRT1 expression in the +Fe control situation compared to Karkar and Douiret (Figure 6B). Thelja and Douiret had a higher HcIRT1 expression level at −Fe versus Karkar (Figure 6B). In summary, the three lines displayed root Fe deficiency response reactions which were most pronounced in case of HcIRT1 induction and medium acidification, while Fe reductase activity increases were not found to be significant. Perhaps the constitutively elevated IRT1 expression level of Thelja is linked with its higher Fe content under −Fe as an adaptation to growth on calcareous soil. ### DISCUSSION Here, we show that the extremophile H. carnosum shows natural variation and phenotypic plasticity with regard to Fe deficiency responses in 8 out of 12 measured parameters. This species is an endemic growing on Tunisian saline and calcareous soil conditions that are known to affect micronutrient use efficiency. Overall, Thelja is the most tolerant isolate showing tolerance to Fe deficiency perhaps as a consequence of its adaptation to calcareous soils. All H. carnosum isolates sensed Fe deficiency and responded to this stress, while the outcome of −Fe stress was different among the ecotypes. The common −Fe symptom elicited by all three lines was the development of a leaf chlorosis. All lines acidified the plant medium and had induced expression of HcIRT1 under −Fe versus +Fe. Several other −Fe symptoms were, however, only displayed by Karkar and Douiret, but not by Thelja. Karkar and Douiret exhibited quite drastic leaf chlorosis at −Fe. This was evident from accelerated leaf chlorosis and the low chlorophyll contents at – versus +Fe after 10 days of −Fe. Leaf chlorosis is a frequently occurring stress symptom in plants since under unfavorable conditions plants tend to reduce photosystem activity by removing chlorophyll and degrading chloroplasts to avoid additional stress caused by the light. This phenomenon can be measured by PAM chlorophyll fluorescence, which was lower in Karkar and Douiret and fits to the leaf chlorosis observations. Moreover, several steps in photosynthetic pigment metabolism and chloroplast ultrastructure are dependent on Fe, which explains the leaf chlorosis in the young expanding leaves after transfer of the plants to −Fe conditions. Fe deficiency resulted in a decrease of Fe contents in Karkar and Douiret expanding leaves, and hence the low Fe status can be regarded as reason for the leaf chlorosis. It is surprising that biomass production was not affected by −Fe in our experiments. We explain this partly by the fact that with ANOVA and Tukey HSD we applied the appropriate but comparably conservative statistical test for multiple comparisons that keeps the family-wise error rate (FWER) at 0.05. Hence, an increasing number of comparisons increases the Type II error (false negative) rate and thus decreases the power for the single comparisons. Perhaps, less conservative tests such as Fisher LSD or two-sample t-tests would have resulted in statistically significant differences. The data indicate that there was a tendency for lower values at – versus +Fe for many parameters even in Thelja, but the differences were not significant according to ANOVA/Tukey HSD. Zn and Cu contents were affected by −Fe in Karkar and Douiret in addition to Fe contents, but not in Thelja. The reduced Fe contents are explained as primary reaction by the low amount of Fe to which the plants were exposed. However, the reduced Zn and Cu contents must have been a secondary reaction of the plant to −Fe. Normally, it would be expected that Zn contents might increase upon −Fe, because increased IRT1 would take up Zn (Li et al., 2014). Perhaps, Hedysarum plants have different capacities to regulate metal homeostasis, and this capacity differs between Thelja, Douiret, and Karkar. Differences in the regulation of Fe reductase activity, IRT1 gene expression and metal contents between different ecotypes were also found for M. truncatula (Li et al., 2014). Under Fe deficiency, plants can suffer from oxidative stress (Ranieri et al., 2001; Zaharieva and Abadia, 2003; Waters et al., 2012; Ramirez et al., 2013). In A. thaliana, the CuSOD (copper/zinc superoxide dismutase) genes CSD1 and CSD2 are induced under Fe deficiency and have been suggested to replace FeSOD's (iron superoxide dismutases) to cope with oxidative stress under iron deficient conditions (Waters et al., 2012). It can be assumed that a similar mechanism exists in leguminous plants and elevated Cu and Zn contents could contribute to the effectiveness of this mechanism. Our observation of higher Cu and Zn levels in Thelja could be one possible explanation for the increased resistance of Thelja to iron deficiency compared to Karkar and Douiret. Hence, the different efficiencies of Cu and Zn uptake under Fe deficiency in Thelja, Karkar, and Douiret could be an important distinctive factor with respect to Fe deficiency tolerance. The stronger −Fe leaf symptoms of Karkar and Douiret suggest that these lines should sense −Fe stress stronger than Thelja. But the two lines did not activate their root Fe mobilization in a stronger manner than Thelja. Karkar reduced more Fe in the root than did Douiret and Thelja. On the other hand, medium acidification capacities were similar between the lines. Thelja and Douiret displayed higher HcIRT1 gene expression than did Karkar. An interesting regulatory phenomenon could be seen for Thelja HcIRT1 gene expression, which was higher at +Fe compared to Karkar and Douiret. One possible explanation is that Thelja might take up more Fe at +Fe than Karkar and Douiret, but store this Fe in the root. Upon Fe deficiency the Fe stores could be remobilized and effectively transported to the shoots. Hence, Thelja could survive better upon −Fe conditions and maintain Fe levels. Since Thelja was collected in a region with calcareous soil condition it is tempting to speculate that the constitutive HcIRT1 expression might contribute to adaptation. On the other hand, Karkar might profit from an inefficient Fe usage, perhaps caused by ineffective internal mobilization and transport in its natural habitat with saline-sodic soil. Some abiotic stress factors induced for example by salt stress affect Fe uptake negatively, which can be explained by the toxicity of metals under water loss and the increased risk of oxidative stress (Le et al., 2016). Several proteins relevant for Fe regulation including IRT1 are controlled at post-translational level (Brumbarova et al., 2015) and hence it would be interesting to combine in the future transcriptomic in comparison to protein studies to assess the physiological activities. The present study focussed on Fe deficiency responses, which are physiologically distinct from responses to calcareous-alkaline medium conditions. Future studies should focus on natural calcareous and saline-calcareous soil conditions. Multiple factors will differ in such experiments, including pH, soil texture, other mineral availabilities and microbial communities. Quite possibly, the ecotypes may differ in their way to mobilize Fe under additional mineral deficiencies and salt stress. Furthermore, the internal iron homeostasis regulation and allocation upon −Fe should gain attention (Schuler et al., 2012). In this respect it is noteworthy that natural variation for Fe efficiency in crops can be manifested at the level of metabolite changes, citrate concentrations for Fe-citrate movement, oxidative stress scavenging and Fe-mobilizing riboflavin patterns (Kabir et al., 2012, 2013, 2015; Jelali et al., 2014; Ben Abdallah et al., 2017). Genome sequence variation of metal homeostasis-relevant genes might account for differences in the gene expression levels or functional SNPs in coding regions. Comparing stress and −Fe responses between young and adult stages as well as under double stress may lead to better understanding of the mechanism of −Fe regulation in this leguminous species. One possibility would be to conduct comparative RNAseq. Gene expression differences between the lines can be used to build novel hypotheses on the physiological mechanisms of tolerance, which could subsequently be validated in biochemical experiments. Our work lies the ground for experiments addressing the mechanistics using the characterized Thelja and Karkar ecotypes as an extreme pair for a detailed natural variation study. ### AUTHOR CONTRIBUTIONS PB, TS, and CA designed the experiments. HBA carried out the experiments. PB, HBA, and HM analyzed the data. CF-S performed the metal determination. PB wrote the manuscript. HBA and HM commented on the manuscript. ### FUNDING This work was supported through an internship and STIBET fellowships from the DAAD and the Tunisian Ministry of Higher Education and Scientific Research (LR10CBBC02). ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01383/ full#supplementary-material ### REFERENCES fpls-09-01383 September 29, 2018 Time: 16:44 # 10 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Ben Abdallah, Mai, Slatni, Fink-Straube, Abdelly and Bauer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. ## Iron and Zinc in the Embryo and Endosperm of Rice (Oryza sativa L.) Seeds in Contrasting 2 0 -Deoxymugineic Acid/Nicotianamine Scenarios ### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil #### Reviewed by: Elsbeth L. Walker, University of Massachusetts Amherst, United States Tracy Punshon, Dartmouth College, United States #### \Correspondence: Ana Álvarez-Fernández [email protected] #### †Present address: Pablo Díaz-Benito, Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Raviraj Banakar, Department of Agronomy, Iowa State University, Ames, IA, United States #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 27 March 2018 Accepted: 25 July 2018 Published: 21 August 2018 #### Citation: Díaz-Benito P, Banakar R, Rodríguez-Menéndez S, Capell T, Pereiro R, Christou P, Abadía J, Fernández B and Álvarez-Fernández A (2018) Iron and Zinc in the Embryo and Endosperm of Rice (Oryza sativa L.) Seeds in Contrasting 2 0 -Deoxymugineic Acid/Nicotianamine Scenarios. Front. Plant Sci. 9:1190. doi: 10.3389/fpls.2018.01190 Pablo Díaz-Benito<sup>1</sup>† , Raviraj Banakar<sup>2</sup>† , Sara Rodríguez-Menéndez<sup>3</sup> , Teresa Capell<sup>2</sup> , Rosario Pereiro<sup>3</sup> , Paul Christou2,4, Javier Abadía<sup>1</sup> , Beatriz Fernández<sup>3</sup> and Ana Álvarez-Fernández<sup>1</sup> \ <sup>1</sup> Department of Plant Nutrition, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Zaragoza, Spain, <sup>2</sup> Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida-Agrotecnio Center, Lleida, Spain, <sup>3</sup> Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Oviedo, Spain, <sup>4</sup> ICREA, Catalan Institute for Research and Advanced Studies, Barcelona, Spain Iron and Zn deficiencies are worldwide nutritional disorders that can be alleviated by increasing the metal concentration of rice (Oryza sativa L.) grains via bio-fortification approaches. The overproduction of the metal chelator nicotianamine (NA) is among the most effective ones, but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative 2<sup>0</sup> -deoxymugineic acid (DMA). The endosperm is the most commonly consumed portion of the rice grain and mediates the transfer of nutrients from vegetative tissues to the metal rich embryo. The impact of contrasting levels of DMA and NA on the metal distribution in the embryo and endosperm of rice seeds has been assessed using wild-type rice and six different transgenic lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb). These transgenic lines outlined three different DMA/NA scenarios: (i) in a first scenario, an enhanced NA level (via overexpression of OsNAS1) would not be fully depleted because of a limited capacity to use NA for DMA synthesis (lack of -or low- expression of HvNAATb), and results in consistent enrichments in NA, DMA, Fe and Zn in the endosperm and NA, DMA and Fe in the embryo; (ii) in a second scenario, an enhanced NA level (via overexpression of OsNAS1) would be depleted by an enhanced capacity to use NA for DMA synthesis (via expression of HvNAATb), and results in enrichments only for DMA and Fe, both in the endosperm and embryo, and (iii) in a third scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (via expression of HvNAATb), and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. Also, quantitative LA-ICP-MS metal map images of the embryo structures show that the first and second scenarios altered local distributions of Fe, and to a lesser extent of Zn. The roles of DMA/NA levels in the transport of Fe and Zn within the embryo are thoroughly discussed. Keywords: metals, laser ablation, ligands, mass spectrometry, rice, seeds ### INTRODUCTION fpls-09-01190 August 18, 2018 Time: 11:57 # 2 The deficiencies of iron (Fe) and zinc (Zn) are among the most important nutritional disorders in plants and humans. These elements play key roles as cofactors and structural components (e.g., Fe in cytochromes and Zn in Zn-finger proteins, respectively) in many proteins. Near 33% of world human population is affected by ferropenic anemia, a low red blood cell count due to Fe deficiency (McLean et al., 2009), whereas Zn deficiency causes about 1.5% of all deaths and about 20% of the perinatal mortality worldwide (Nriagu, 2007). Furthermore, a potential outcome of both metal deficiencies is neuropsychological impairment (Sandstead, 2000). Many of these cases of malnutrition could be solved with a diet enriched in Fe and Zn (De Benoist et al., 2008; White and Broadley, 2009). Foods rich in micronutrients such as meat and vegetables, unlike staple foods, are expensive and cannot be stored for long periods. Since rice is a staple food in large areas of the world, particularly in underdeveloped regions, biofortification of rice grains with Fe and Zn is a realistic target to alleviate these nutritional disorders. In most parts of the world, rice is traditionally cooked after milling and polishing, reducing the nutritional value because of the removal of the metal-rich bran and embryo, with only the endosperm remaining. Also, rice can be treated hydrothermally (parboiling) prior to milling to reduce breakage, increasing the nutritional value because of micronutrient transport from bran to endosperm, although changes of color, odor and texture, as well as mycotoxin risks, may arise (Rohman et al., 2014). Conventional, agronomic and transgenic approaches have been used for rice biofortification (reviewed by Garg et al., 2018), with approaches based on molecular genetics having the advantage that any gene with a demonstrated utility may be further used for targeting the biofortification of other crops. Furthermore, whereas diet diversification might be an option in principle, in practical terms poor people in developing counties cannot afford a diverse diet. Therefore, a major challenge for biofortification strategies in rice is to increase the concentrations of Fe and Zn in the endosperm. Rice plants take up Fe from the soil using mechanisms classically ascribed to Strategies I and II (Ishimaru et al., 2006). Strategy I, used by non-graminaceous species, involves the uptake of Fe(II) via a Fe-Regulated Transporter (IRT) (Vert et al., 2002). Rice roots do express OsIRT1 and this transporter is strongly upregulated upon Fe deficiency (Ishimaru et al., 2006). In Strategy II, used by Gramineae, Fe acquisition is mediated by the synthesis and secretion of phytosiderophores (PSs) of the mugineic acid family (MAs) (Kobayashi et al., 2006). The synthesis of MAs starts from the condensation of three S-adenosyl methionine molecules to produce nicotianamine (NA) via nicotianamine synthase (NAS). Then, 2<sup>0</sup> -deoxymugineic acid (DMA) is synthesized from NA via nicotianamine aminotransferase (NAAT) and DMA synthase. In response to Fe deficiency, rice roots synthesize DMA (Takagi, 1993), which is secreted to the rhizosphere via TOM1 (Transporter Of Mugineic acid 1) (Nozoye et al., 2011). The secreted DMA is able to solubilize sparingly soluble Fe(III) by forming Fe(III)-DMA complexes, which are then taken up by root cells via transporters of the YSL (Yellow Stripe 1-Like) family (Curie et al., 2009). Zinc is usually taken up by plants as the free Zn(II) ion by root epidermal cells (Ishimaru et al., 2011; Sinclair and Krämer, 2012). Also, PSs can form Zn complexes that are as stable as Fe(III)-PS (Murakami et al., 1989), and both the secretion of PS and uptake of Zn-PS via YSL transporters have been observed in grasses (von Wirén et al., 1996; Suzuki et al., 2006; Widodo et al., 2010). The short- and long-distance transport of Fe and Zn in grasses occurs both as free ions and metal complexes. Different PS-metal complexes have been found in plant fluids, including Fe(III)-DMA and Zn(II)-DMA in the xylem sap of wheat (Xuan et al., 2006), and Fe(III)-DMA and Zn(II)-NA in the phloem sap of rice (Nishiyama et al., 2012). Since both plant fluids transport nutrients from maternal to filial tissues at the reproductive stage (Waters and Grusak, 2007), Fe and Zn in grains can originate either from a direct root-to-seed route via xylem or from the remobilization of Fe from old and senescing leaves via phloem (Grillet et al., 2014; Yoneyama et al., 2015). An internal transport of these metals also occurs once they are in the grain, since the developing embryo is a sink for nutrients and the endosperm constitutes a nutrient reservoir. A complex network of transporters belonging to different families mediates the movement of both metals within cells and the whole plant; some proteins (e.g., IRT, P1B-type heavy metal ATPases) are capable of transporting Zn and Fe divalent ions (Takahashi et al., 2012; Kolaj-Robin et al., 2015), whereas others are capable of transporting Zn and Fe complexes (e.g., YSL family transport metal-NA or metal-PSs complexes; Curie et al., 2009). The fact that mutations in the genes involved in NA and DMA synthesis and those of YSL transporters do not cause substantial decreases in the seed Fe concentrations (e.g., osnas3, Lee et al., 2009b) supports that metal transport systems in plants are redundant. On the other hand, the fact that the overexpression of genes involved in DMA/NA synthesis and those of YSL transporters only lead to limited increases in seed Fe concentrations (Banakar et al., 2017a,b) support the existence of regulatory feedback loops. Once in the rice grain, Fe may be stored in ferritins to avoid toxicity (Briat et al., 2010), sequestered in vacuoles (Kim et al., 2006) or bound to phytate, a P-rich molecule preferentially located in the aleurone layer (Persson et al., 2009). Phytate is also considered to control Zn levels in seeds (Raboy, 2003), and has a low bioavailability during human digestion (Guttieri et al., 2004; Gupta et al., 2015). A range of transgenic approaches have been used to increase micronutrient concentrations in rice grains (Bashir et al., 2013; Nozoye, 2018), including: (i) increases in the expression of NA, DMA, YSL and ferritin synthesis genes; (ii) increases in absorption promoters, and/or (iii) decreases in inhibitors of absorption in human gut. The highest increase achieved so far in the concentrations of Fe and Zn in polished seeds has been achieved with an OsNAS2-SoyferH-1 construct, leading to 6- and 4-fold increases, respectively, over the WT values (Trijatmiko et al., 2016). Several studies have found a positive effect of increasing NA synthesis in achieving rice biofortification with Fe and Zn (Nozoye, 2018), but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative DMA. Moreover, most previous studies have focused on the grain endosperm, with the embryo, a part of the seed of outmost importance for seed formation, germination and viability, being studied in less detail. In this work, the impact of contrasting levels of NA and DMA on the distribution of metals in the embryo and endosperm of rice seeds has been studied, using wild-type (WT) rice and six transgenic lines overexpressing OsNAS1 and/or expressing barley NAAT (HvNAATb). Increasing only DMA led to Fe enrichments in the embryo and endosperm, whereas increasing DMA in combination with NA produced Fe and Zn enrichments in both tissues. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was also used to determine the spatial localization and concentrations of Fe, Zn and other elements within the embryo structures, providing the first quantitative set of data for these metals in the embryo tissues of biofortified rice seeds. We discuss the changes induced in the elemental distribution in contrasting DMA/NA scenarios, providing new insights into the Fe and Zn transport mechanisms within the embryo. ### MATERIALS AND METHODS ### Plant Material Rice plants (Oryza sativa L. cv EYI 105) were transformed to obtain genotypes overexpressing OsNAS1 and/or HvNAATb genes and therefore with high levels of NA and/or DMA. The details of the cloning, expression and transformation were described in detail in Banakar et al. (2017b). The six lines used are two overexpressing OsNAS1 alone (N1 and N2), two expressing HvNAATb alone (D1 and D2) and two expressing OsNAS1 and HvNAATb together (ND1 and ND2). Lines ND1 and ND2 were also used, although in different growth conditions, in Banakar et al. (2017b). ### Gene Cloning The cDNAs of OsNAS1 (GenBank ID: AB021746.2, 999 bp) and/or HvNAATb (GenBank ID: AB005788.1, 1,656 bp) were cloned from roots of rice (O. sativa cv EYI 105) and barley (Hordeum vulgare L. cv Ordalie) grown in vitro on MS medium without Fe (Murashige and Skoog, 1962) for 2 weeks. Total RNA was extracted with RNeasy Plant Mini kit (Qiagen, Hilden, Germany) and 1 µg of RNA was used for reverse transcription using Omniscript RT Kit (Qiagen) by RT-PCR. The full-size cDNAs for OsNAS1 (999 bp) and HvNAATb (1,656 bp) were amplified by PCR using the primer combinations OsNAS1-BamHI-FOR (5<sup>0</sup> -AGG ATC CAT GGA GGC TCA GAA CCA AGA GGT CG-3<sup>0</sup> ) and OsNAS1-HindIII-REV (5<sup>0</sup> -AAA GCT TCA TAA TAT AGT GCG CCT GAT CGT CCG GCT GT-3<sup>0</sup> ), and HvNAATb-BamHI-FOR (5<sup>0</sup> -AGG ATC CAT GGC CAC CGT ACG GCC AGA GAG CGA CG-3<sup>0</sup> ) and HvNAATb-HindIII-REV (5<sup>0</sup> - AAA GCT TCT AGC AAT CAT CGC TCG CTC GAA TTT CTC-3<sup>0</sup> ), respectively. The products were transferred to the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA) for sequencing and verification. The OsNAS1 and HvNAATb cDNAs were inserted at the BamHI and HindIII sites of expression vector pAL76 (Christensen and Quail, 1996), which contains the maize UBI1 promoter and first intron, and an Agrobacterium tumefaciens nos transcriptional terminator. A separate vector harboring the constitutive cauliflower mosaic virus 35S promoter (CaMV35S) was used to provide the hygromycin phosphotransferase (hpt) selectable marker (Christou and Ford, 1995). ### Rice Transformation and Growth Conditions Rice embryos were isolated from Oryza sativa L. (cv EYI 105) mature seeds and grown on MS medium containing 2.5 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D) as in Sudhakar et al. (1998). After 7 days, embryos were incubated on highosmoticum MS medium (0.2 M mannitol, 2.5 mg L−<sup>1</sup> 2,4- D) for 4 h (Sudhakar et al., 1998; Valdez et al., 1998), and then bombarded with gold particles carrying the transgenes and the hpt selectable marker (Christou et al., 1991). Bombarded embryos were selected on MS medium supplemented with 2.5 mg L−<sup>1</sup> 2,4-D and 30 mg L−<sup>1</sup> hygromycin, and callus pieces were transferred sequentially to shooting and rooting medium containing hygromycin (Sudhakar et al., 1998; Valdez et al., 1998). Regenerated plantlets were transferred to pots filled with substrate (Traysubstract, Klasmann-Deilmann GmbH, Geeste, Germany) and grown flooded in large trays in growth chambers at 26◦C, 900 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PPFD PAR with a 12/12 h light/dark regime and 80% relative humidity. Plants were watered with 100 µM Fe(III)-EDDHA (Sequestrene 138 Fe G-100; Syngenta Agro SA, Madrid, Spain) until flowering, and then self-pollinated. The Fe(III)-EDDHA solution in the trays was replaced every week. T<sup>0</sup> plants were grown to maturity, T<sup>1</sup> seeds were harvested and the resulting plants were crossed over two generations to generate a homozygous T<sup>3</sup> population. T<sup>3</sup> seeds from the transgenic lines were germinated on <sup>1</sup>/<sup>2</sup> MS medium containing 30 mg L−<sup>1</sup> hygromycin, whereas WT seeds were germinated on <sup>1</sup>/<sup>2</sup> MS medium without hygromycin. Five-day-old seedlings from wild type and transgenic lines were transferred to pots filled with substrate as described above, and maintained until the T<sup>4</sup> seeds had matured. Sampling of the T<sup>3</sup> flag leaf was performed to confirm the expression of the genes of interest and T<sup>4</sup> seeds were harvested to study the metal quantitative distribution in different seed tissues. The anatomical denominations in this study comply with the monograph by Hoshikawa (1989). Grains from the same panicle were harvested, and some of them were stored at 4◦C until metal localization analyses and others de-husked by hand, to avoid metal contamination from the de-husking machine. Then, brown seeds were separated into embryo and endosperm, in both cases maintaining the corresponding aleurone layer, using new stainless steel razor blades and binocular magnifying glasses. For each genotype, embryo and endosperm samples were obtained pooling materials from 50–100 and 10 seeds, respectively, and 3– 4 replications were used. Samples were ground in liquid N<sup>2</sup> with ceramic mortar and pestle until a fine powder was obtained, and aliquots were stored at −80◦C until analysis. ### RNA Blot Analysis fpls-09-01190 August 18, 2018 Time: 11:57 # 4 Total RNA was isolated from the flag leaf (T<sup>3</sup> generation) using the RNeasy Plant Mini Kit (Qiagen), and 20-µg aliquots were fractionated on a denaturing 1.2% agarose gel containing formaldehyde before blotting. The membranes were probed with digoxigenin-labeled partial OsNAS1 or HvNAATb cDNAs at 50◦C overnight, using DIG Easy Hyb (Roche Diagnostics, Mannheim, Germany). After washing and immunological detection with anti-DIG-AP (Roche Diagnostics) according to the manufacturer's instructions, CSPD chemiluminescence (Roche Diagnostics) was detected on Kodak BioMax light film (Sigma–Aldrich, St Louis, MO, United States). #### Analysis of Metals, Nicotianamine and 2 0 -Deoxymugineic Acid in the Embryo and Endosperm Fifty mg of ground embryo or endosperm tissue from grains of the same panicle were dried at 60◦C and digested with ultrapure 21% HNO<sup>3</sup> (Trace Select Ultra, Fluka) and 6% H2O<sup>2</sup> (Suprapur, Merck) for 55 min at 190◦C in an Ethos 1 microwave oven (Milestone Srl., Sorisole, Italy). Digests were analyzed (3 independent replicates) for Fe, Mn, Cu, and Zn by inductively coupled plasma mass spectrometry (ICP-MS), using an Agilent 7500ce (Agilent, Santa Clara, CA, United States) and monoelemental standard solutions for ICP-MS (Inorganic Ventures, Christiansburg, VA, United States). Recoveries were 98.3, 95.8, 97.0, and 95.0% for Fe, Mn Cu, and Zn, respectively, and limits of detection were 20, 2, 2 and 20 µg L−<sup>1</sup> for Fe, Mn Cu, and Zn, respectively. Concentrations are expressed as µg metal g tissue DW−<sup>1</sup> . Nicotianamine and DMA were extracted from 50 mg of ground embryo or endosperm tissue (3–4 independent replicates) with 300 µL Type I water containing 18 µL of 1 mM nicotyllysine -used as internal standard- following the procedure previously developed for rice seeds (Banakar et al., 2017a). The extracts were analyzed for NA and DMA using an Alliance 2795 HPLC system (Waters, Mildford, MA, United States) coupled to a time-of-flight mass spectrometer (MS-TOF; MicrOTOF, Bruker Daltonics, Bremen, Germany) equipped with an electrospray (ESI) source. For a detailed description of the method, see Banakar et al. (2017a). The limit of quantification (LOQ), defined as the concentration giving a signal to noise ratio of 10, was 1.0 µg g −1 tissue FW for NA and DMA. All ligand concentrations are expressed as µg NA (or DMA) g tissue FW−<sup>1</sup> . ### Imaging Elemental Distribution in Seed Sections ### Sample Preparation Thin sections (50–70 µm-thick) were obtained from fully developed, de-husked rice grains of the different genotypes using a vibrating blade microtome (VT1000 S, Leica Microsystems GmbH, Wetzlar, Germany), following the protocol described by Johnson et al. (2011). Seeds were glued (with Loctite Super Glue-3, Barcelona, Spain) to the excised bottom of a 1.5 mL plastic Eppendorf tube, and blades used were Chrome Platinum (Bic, Clichy, France). Vibratome parameters were a blade movement speed of 0.2 mm s−<sup>1</sup> and a vibration frequency of 70 Hz. A 100 µm-thick Kapton polyimide film (DuPont, Des Moines, IA, United States) was used to hold the tissue section as cutting proceeded, to minimize endosperm fragmentation (Johnson et al., 2011). Longitudinal dorso-ventral seed sections were used for optical microscopy, Perl's staining and LA-ICP-MS analysis. Sections were transferred to synchrotron adhesive tape (Leica), attached to glass slides, observed with a stereomicroscope (MZ16, Leica) and images taken with the Leica Application Suite V3.5. Sections were stored at 4◦C until LA-ICP-MS analysis. ### Perl's Prussian Blue Staining Seed sections (60 µm-thick) were used immediately to localize Fe using Perls staining. Sections were incubated with a solution containing 2% K4[Fe(CN)6] and 2% HCl for 15 min at room temperature. This staining technique allows for the detection of labile Fe forms, including Fe complexes with NA and citrate, Fe hydroxides and inorganic Fe, as the blue compound ferric ferrocyanide (Roschzttardtz et al., 2009; Rios et al., 2016). Stained sections were observed with a stereomicroscope (MZ16, Leica) and images taken with the Leica Application Suite V3.5. ### LA-ICP-MS Analysis Rice seed sections (60 µm-thick) adjacent to those used for Perl's staining were placed on synchrotron adhesive tape (Leica) and directly analyzed using a Laser Ablation (LA) system (LSX-213, Teledyne Cetac Technologies, Omaha, NE, United States) coupled to an ICP-MS instrument (Element II, Thermo Fischer Scientific, Waltham, MA, United States). Preliminary analyses were first carried out by driving LA straight lines through whole longitudinal dorso-ventral seed sections, and intense ICP-MS signals for the elements of interest were observed only in the embryo, with the endosperm providing very low or no signal. Optimized LA settings allowed for distinguishing embryo structures and obtaining signals of good intensity for the different elements, but the analysis time required for a single whole seed section was longer than 15 h. Therefore, the LA-ICP-MS analyses had to be restricted to the embryo and neighboring endosperm. Optimized settings used for the LA-ICP-MS analysis (e.g., laser spot diameter and scan speed) are shown in Table 1. The net intensity of the signal obtained for each element ( <sup>31</sup>P, <sup>32</sup>S, <sup>55</sup>Mn, <sup>56</sup>Fe, <sup>63</sup>Cu, <sup>64</sup>Zn) was normalized using that of <sup>13</sup>C as an internal standard for quantification purposes. Quantification of the selected elements was carried out using two certified reference materials (CRMs) for calibration: the rice flour standards NIST 1568b (National Institute of Standards and Technology, United States) and NCS ZC73028 (LGC Standards, UK). Powdered CRMs were pressed to pellets 5 mm in diameter using a laboratory press (applying 2 t for 5 min) and subsequently analyzed using the same experimental conditions optimized for the seed sections. Three ablation lines were performed for each CRM along the pellet, and the resulting normalized intensity signals, together with respective elemental concentrations were used to build calibration curves. The linear regression equations and coefficients for <sup>31</sup>P, <sup>32</sup>S, <sup>55</sup>Mn, <sup>56</sup>Fe, <sup>63</sup>Cu, <sup>64</sup>Zn calibration curves are shown in Supplementary Table S1 and the calibration curve obtained for <sup>32</sup>S is shown as an example in Supplementary Figure S1. Quantitative two-dimensional images of the elemental distributions in seed sections were created using the software packages Origin <sup>R</sup> (OriginLab, Northampton, MA, United States) and ImageJ (NIH, Bethesda, WA, United States). Each data point (or pixel) was converted from intensities into concentrations, using the calibration curve for each element of interest. Then, quantitative elemental map images were first obtained by processing concentration data with the software Origin <sup>R</sup> . Secondly, the concentration data for Fe and Zn were also processed with the software ImageJ <sup>R</sup> , which permits obtaining images with a higher resolution; these maps were obtained for the WT and one line each from the three transgenic types. In the images processed with ImageJ, different concentration scales were used for each section to highlight differences in metal localization and concentration along the seed structures. Limits of detection (LODs) were calculated by using the 3s criterion (3sb/S), where s<sup>b</sup> is the standard deviation of 5 independent measurements of the blank value in counts per s and S is the sensitivity for the corresponding analyte isotope obtained by measuring the CRM NIST 1568b. At the selected operating conditions and using the CRM pellets, LODs were (in µg g−<sup>1</sup> ) 0.77 for <sup>31</sup>P, 11 for <sup>32</sup>S, 0.10 for55Mn, 0.62 for <sup>56</sup>Fe, 0.049 for <sup>63</sup>Cu, and 0.13 for <sup>64</sup>Zn, with the higher LOD for S being due to the relatively high background. The detection limits in the rice seed sections were higher, likely due to differences between the thin seed sections and the pressed pellets used for rice flour standards. ### Statistical Analysis Statistical analysis was carried out with SPSS Statistics (v.22, IBM, New York, NY, United States) using (i) ANOVA test (P ≤ 0.05) to determine differences between data from transgenic and WT plants and (ii) bivariate Pearson correlation to determine whether significant linear relationships existed between the FIGURE 1 \| RNA blot analysis showing transgene expression in the leaf tissue of wild-type (WT) and six independent transgenic lines, two expressing OsNAS1 (lines N1 and N2), two expressing HvNAATb (lines D1 and D2) and two co-expressing OsNAS1 and HvNAATb (lines ND1 and ND2). rRNA, ribosomal RNA. Plants were grown under nutrient-sufficient conditions, and total RNA was isolated from flag leaves at physiological maturity. concentrations of metal ligands and metals in the embryo and endosperm. ### RESULTS We co-transformed rice mature seed-derived embryos with separate constructs harboring rice NAS (OsNAS1) and/or barley NAAT (HvNAATb), along with the selectable marker hpt. RNA blot analysis using rRNA isolated from flag leaf tissue identified lines expressing OsNAS1 alone (N1 and N2), HvNAATb alone (D1 and D2) and OsNAS1 and HvNAATb together (ND1 and ND2) (Figure 1). There was considerable variation in the transgene expression levels among the lines used: the expression of OsNAS1 was higher in N1 than in N2, the expression of HvNAATb was much higher in D2 than in D1, and the expression of both OsNAS1 and HvNAATb were higher in ND2 than in ND1. Brown seeds from these plants were harvested (T<sup>4</sup> seeds), separated into embryo and endosperm, in both cases including their corresponding aleurone layer, and analyzed for NA, DMA and metals. ### Nicotianamine and 2<sup>0</sup> -Deoxymugineic Acid Concentrations in the Embryo and Endosperm The embryo and endosperm of the WT and transgenic lines were analyzed by HPLC-ESI-MS to determine the concentrations of NA and DMA (Figure 2). This study is, to the best of our knowledge, the first to report NA and DMA concentrations in the rice embryo, since previous studies have only used polished and/or unpolished seeds, without analyzing embryos. In WT seeds, the NA concentration was 6.0 ± 1.5 µg g−<sup>1</sup> FW in the embryo and below the LOQ (marked as BLQ in Figure 2) in the endosperm, whereas the DMA concentrations were 24.2 ± 1.4 and ND2). Plants (WT and T<sup>3</sup> transgenic lines) were grown under nutrient-sufficient conditions, and the WT and T<sup>4</sup> seeds were harvested at physiological maturity. Asterisks indicate significant differences between WT and transgenic plants as determined by Student's t-test (P < 0.05). Values shown are means ± SE, n = 3–4. BLQ: below limit of quantification. and 13.5 ± 1.0 µg g−<sup>1</sup> in the embryo and endosperm, respectively (Figure 2). Therefore, the embryo was richer in NA and DMA than the endosperm; the DMA/NA ratios were approximately 4 and very high in the embryo and endosperm, respectively (Supplementary Figure S2). DMA was also reported to be more abundant than NA in other rice compartments, including seeds (polished and unpolished; Masuda et al., 2009; Lee et al., 2011; Masuda et al., 2012; Trijatmiko et al., 2016), roots and leaves (Masuda et al., 2009), as well as phloem and xylem saps (Kato et al., 2010; Ando et al., 2013). In transgenic lines, changes in NA and DMA concentrations in the embryo and the endosperm were observed when compared to the WT (Figure 2). The embryo NA concentrations of lines N1, N2 and ND1 were 15-, 2.5-, and 3.0-fold higher, respectively, whereas endosperm NA concentrations were well above the LOQ (29.1 ± 4.8, 6.4 ± 0.1, and 16.8 ± 0.3 µg g−<sup>1</sup> , respectively). In contrast, in lines D1, D2 and ND2, the embryo NA concentrations were lower than those of the WT (although not significantly at P ≤ 0.05). The embryo DMA concentrations of lines N1, D1, ND1 and ND2 were 3.5-, 4.2-, 4.1-, and 12 fold higher, respectively, than those in the WT. The endosperm DMA concentrations in lines N1, N2, ND1 and ND2 were also much higher than those in the WT (10.5-, 6.2-, 9.2-, and 19.0-fold, respectively), whereas they increased 2-fold in D2 and decreased by 71% in D1. As a result of these changes, the DMA/NA ratios in the embryo of N1 and N2 (1 and 3, respectively) were lower than those in the WT (Supplementary Figure S2). Conversely, in D1, D2, ND1, and ND2 the DMA/NA ratios in the embryos were higher than in the WT, 34, 10, 7 and 185, respectively. On the other hand, the DMA/NA ratios in the endosperm were very high in D1, D2, and ND2 (in these three lines the NA concentrations were below the LOQ), whereas in N1, N2 and ND1 they were much lower, 5, 13 and 7, respectively. ### Metal Micronutrient Concentrations in the Embryo and Endosperm The embryo and endosperm of the WT and transgenic lines were analyzed by ICP-MS to determine the concentrations of Fe, Zn, Mn, and Cu (Figure 3). In the WT, the Fe concentrations were 99 ± 6 and 20 ± 1 µg g−<sup>1</sup> DW in the embryo and endosperm, respectively. In lines N1, N2, ND2, and ND1 the Fe concentrations were significantly higher both in the embryo (1.3 to 2.1-fold increases over the WT values) and the endosperm (1.7- to 2.9-fold increases). In contrast, in D1 and D2 the Fe concentrations in the embryo and endosperm were not affected significantly, with the exception of a 33% decrease in the endosperm of D1. The Zn concentrations in the WT were 102 ± 2 and 21 ± 1 µg g −1 in the embryo and endosperm, respectively (Figure 3). In the embryo, Zn concentrations increased significantly over the WT values in N1 (1.3-fold) and N2 (1.2-fold), whereas they decreased by 40% in ND2. In the endosperm, Zn concentrations increased over the WT values in N1 (2.5-fold), N2 (1.8-fold), and ND1 (2.5 fold). The Mn concentrations in the WT were 76 ± 1 and 16 ± 2 µg g −1 in the embryo and endosperm, respectively (Figure 3). Significant changes in the Mn concentration in the embryo were only observed in N2, ND1 (a 18% decrease in both lines) and D1 (a 1.3-fold increase). In the endosperm, the only significant change in Mn concentration was found in N1, which showed a 1.4-fold increase when compared to the WT. The Cu concentrations in the WT were 10 ± 1 and 7 ± 1 µg g −1 in the embryo and endosperm, respectively (Figure 3). In the embryo, the only significant changes in Cu concentrations were in N2 (a 1.3-fold increase), and ND1 and ND2 (2-fold increases). In the endosperm, the only changes found in Cu concentrations were in N1 (a 27% decrease), and N2 and ND1 (1.2 and 1.4-fold increases, respectively). ### Correlations Between NA, DMA and Metal Micronutrients in the Embryo and Endosperm The relationships between the concentrations of metal ligands and metals in the embryo and endosperm were studied by bivariate Pearson correlation analysis, using all data (in nmol g <sup>−</sup><sup>1</sup> DW) from the WT and transgenic lines (Supplementary Table S2). Correlations found were different in the endosperm and embryo. In the embryo, DMA was positively correlated with Cu (r = 0.610; P ≤ 0.05), whereas NA was positively correlated with Fe and Zn (r = 0.643 and 0.647, respectively; in both cases significant at P ≤ 0.05). In the endosperm, there were highly significant and strong positive correlations between DMA and Fe (r = 0.702; P ≤ 0.01) and DMA and NA (r = 0.943; P ≤ 0.01), whereas no significant correlation was found between DMA and other metals (Supplementary Table S2). There was no correlation between NA and metals, whereas several correlations between metals were found, including Mn vs. Zn (r = 0.782; P ≤ 0.01), Mn vs. Cu (r = 0.725; P ≤ 0.01) and Fe vs. Zn (r = 0.566; P ≤ 0.05). Correlations between Fe and Zn have been found in previous studies (Lombi et al., 2011; Anuradha et al., 2012; Banakar et al., 2017b; Kampuang et al., 2017), and are expected due to the fact that both metals share mechanisms for uptake, short- and long-distance transport in the plant and intracellular trafficking. In some cases, parameters measured in the embryo showed correlations with those measured in the endosperm (Supplementary Table S2). There were strong positive correlations between NA in the endosperm and DMA in the embryo (r = 0.986; P ≤ 0.01), and negative correlations between endosperm Fe and Cu, and embryo Cu (r = −0.644 and −0.604, respectively; in both cases at P ≤ 0.05). ### Perl's Staining of Rice Seed Sections The Perl's staining of longitudinal dorso-ventral sections of WT and transgenic seeds reveal the accumulation of Fe in the embryo region and the aleurone layer, whereas the blue color was absent in the endosperm (an optical image of the WT seed is shown in Figure 4A, and the Perl's stain is shown in Figure 4B). The distribution of Fe in the embryo differed among genotypes studied. In the WT, Fe was accumulated mainly in the epithelium, and at lower levels in the scutellum and some parts of the aleurone layer. In N1 (overexpressing only OsNAS1) the whole embryo had more labile Fe than the WT. This increase in labile Fe was marked in the root primordia and scutellum, and also visible in the epithelium, the tip of the leaf primordia and the aleurone layer that covers the embryo and endosperm. While the pattern of Fe over-accumulation was similar among lines overexpressing OsNAS1, in ND2 (co-expressing OsNAS1 and HvNAATb) there was a much higher Perl's stain than in N1, when OsNAS1 was overexpressed alone. In D2 (expressing only HvNAATb) there was an over-accumulation of labile Fe in specific areas of the embryo, mainly in the root primordia and scutellum, although this effect was much less intense than in N1, whereas the Perl's stain in the epithelium and aleurone layer did not differ from the WT. ### Quantitative Images of the Elemental Distributions in Rice Seed Sections Obtained by LA-ICP-MS The quantitative two-dimensional images obtained for Fe, Zn, Mn, Cu, P and S distribution in the seeds are shown in Figures 5, 6, with the lowest and highest concentrations being represented in dark blue and red, respectively. First, maps are presented together with the corresponding optical images of the same sections, using the same scale for all genotypes (Figure 5). The match between elemental maps and optical images allows for the allocation of elemental concentrations to the different embryo structures. Also, high resolution maps were drawn using different concentration scales for each sample to depict optimal contrast (Figure 6). This is the first time quantitative LA-ICP-MS elemental map images (Fe, Zn, Mn, Cu, P, and S) have been obtained for the embryo structures of WT and biofortified rice seeds. Previous studies applying LA-ICP-MS imaging to rice seeds did not provide quantitative images for Fe, and those for Zn had only a low resolution (Wirth et al., 2009; Basnet et al., 2014, 2016). The first remarkable aspect in the elemental distribution was the preferential accumulation of most elements in the embryo, with the element concentrations in the endosperm being generally lower, with the only exceptions of Cu and S (Figure 5). This is in agreement with previous results reported by using semiquantitative, high-resolution techniques such as synchrotronbased X-ray fluorescence spectroscopy (Lombi et al., 2009; Takahashi et al., 2009; Wirth et al., 2009; Johnson et al., 2011; Iwai et al., 2012; Lu et al., 2013; Kyriacou et al., 2014) and secondary ion mass spectrometry (Kyriacou et al., 2014). Since most seed parts are quite heterogeneous, their composition are best described in terms of concentration ranges for each element. g g lines) were grown under nutrient-sufficient conditions. WT and T<sup>4</sup> seeds were harvested at physiological maturity. In the case of Fe, there were large differences in concentrations between seed parts, with the distribution being quite different in the WT and some of the transgenic lines, with the exception of the endosperm, where Fe concentrations were below 10 µg Fe g−<sup>1</sup> for all genotypes (Figures 5, 6). In the WT, the highest Fe concentrations were found in the epithelium and root primordium (70–300 µg Fe g−<sup>1</sup> ), whereas lower concentrations were found in the aleurone layer (10–140 µg Fe g−<sup>1</sup> ), leaf primordium (25–100 µg Fe g−<sup>1</sup> ) and scutellum (25–75 µg Fe −1 ) (Figures 5, 6). In N1 and N2, the Fe concentration was increased in the scutellum (to the range 150–650 µg g−<sup>1</sup> in both lines), leaf primordium (150–950 and 150–350 µg g−<sup>1</sup> in N1 and N2, respectively), root primordium (150–400 and 150–600 µg −1 in N1 and N2, respectively) and aleurone layer (70–300 µg Fe g−<sup>1</sup> in both lines) (Figure 5; see also N2 in Figure 6). Lines D1 and D2 showed lower Fe concentrations in most embryo tissues than those found in the WT, with D1 being more affected than D2 (Figure 5; see also D2 in Figure 6). Iron concentrations decreased in D1 and D2 in the scutellum (below 50 µg g−<sup>1</sup> ) and HvNAATb (lines ND1 and ND2). Plants (WT and T<sup>3</sup> transgenic lines) were grown under nutrient-sufficient conditions, and the WT and T<sup>4</sup> seeds were harvested at physiological maturity and dehusked. 60 µm-thick seed sections were used for LA-ICP-MS analysis. Elemental images were obtained processing LA-ICP-MS data with the software Origin <sup>R</sup> . Color scales represent the concentrations for each element, with the lowest ones in dark blue and the highest ones in red. Depending on the element, the scale bar indicates the elemental concentrations in µg g−<sup>1</sup> (Fe, Zn, Mn, and Cu) and in mass fraction percentage (S and P). For a given element, the same scale was used in all genotypes. Pictures shown in the first row are optical images of the sections subjected to LA-ICP-MS analysis. AL, aleurone layer; LP, leaf primordium; RP, root primordium; SC, scutellum; SE, starchy endosperm; EP, epithelium. the root primordium (<50 µg g−<sup>1</sup> and to 50–100 µg g−<sup>1</sup> in D1 and D2, respectively), whereas decreased in the leaf primordium and epithelium only in D1 (<50 and 50–100 µg g−<sup>1</sup> , respectively) and increased in the aleurone layer only in D1 (50–250 µg g−<sup>1</sup> ). Lines ND1 and ND2 showed large Fe concentration increases in the epithelium (200–900 µg g−<sup>1</sup> ; Figure 5; see also ND1 in Figure 6), compared to the values found in the WT and the other four transgenic lines. Other relevant changes in Fe distribution in ND1 and ND2 were: (i) increases in root primordium Fe concentrations over those in the WT but not always over those in N2 (up to 200–900 and 100–400 µg g−<sup>1</sup> , respectively), (ii) increases in scutellum Fe concentrations over those in the WT but only slightly higher than those in N1 and N2 (150–550 and 100–350 µg g−<sup>1</sup> , respectively), and (iii) decreases in the leaf primordium Fe concentrations (<50 µg g−<sup>1</sup> ) below those in the WT. In the case of Zn, large differences in concentrations were also observed between the different seed parts, with modifications in the distribution in the transgenic genotypes when compared to the WT, whereas Zn could not be detected in the endosperm of any genotype (Figures 5, 6). The WT seed showed the highest Zn concentration in the leaf primordium (500–1100 µg g−<sup>1</sup> ), followed by the scutellum and epithelium (50–200 µg g−<sup>1</sup> ) and the aleurone layer (<100 µg g−<sup>1</sup> ). Lines N1 and N2 showed lower concentrations of Zn than those found in the WT in the leaf primordium (300–800 and 50–1100 µg g−<sup>1</sup> , respectively), whereas in the root primordium, scutellum, epithelium and aleurone layer the Zn concentrations increased only in N2 (50– 1500, 200–550, <500 and <200 µg g−<sup>1</sup> , respectively) when compared with the WT. In D1 and D2, Zn concentrations in both the leaf primordia (<600 and 200–500 µg g−<sup>1</sup> , respectively) and scutellum (50–100 and <75 µg g−<sup>1</sup> , respectively) were lower than those in the WT. Other changes in Zn distribution when compared with the WT were different for these two lines: Zn increased in D1 in the root primordia and epithelium (500–1000 and 150–350 µg g−<sup>1</sup> , respectively) and decreased in D2 (<75 µg g −1 in both tissues). In ND1 and ND2 Zn concentrations in the leaf primordia (300–700 µg Zn g−<sup>1</sup> , respectively) were lower than those in the WT, and similar to those found in N1 and N2. Also, moderate increases in the Zn concentrations in the root primordium and scutellum were observed in the two double transgenic lines, especially when compared with those in N1. Manganese could not be detected in the endosperm of any genotype, and was generally localized in high concentrations in the leaf and root primordia in all genotypes (100–400 and 100– 130 µg g−<sup>1</sup> , respectively), with concentrations in the epithelium and aleurone layer being also similar (<40 and 50–150 µg g−<sup>1</sup> , respectively; Figure 5). The only exception was ND1, which showed higher Mn concentrations in all tissues (100–500 µg Mn g −1 ; Figure 5). Copper was the less abundant micronutrient of those investigated, and differences in distribution between lines were less marked (Figure 5). In the WT, this element was mostly found in the aleurone layer, scutellum and leaf primordium (concentrations in the range of 5–15 µg Cu g−<sup>1</sup> ), with Cu concentrations in the rest of the tissues being ≤10 µg g−<sup>1</sup> , with the exception of the inner endosperm, where Cu could not be detected. In N1, N2, ND1 and ND2 some changes in Cu distribution were observed: increases in Cu concentrations in the root primordia (9–12, 8–25, 10–30 µg g−<sup>1</sup> in N1, ND1 and ND2), leaf primordia (9–40 µg g−<sup>1</sup> in N1), and aleurone layer (10–30, 8–25, 8–25 µg g−<sup>1</sup> in N2, ND1 and ND2). In contrast, in D1 and D2 no changes were observed in the Cu distribution. Phosphorus was present with similar distribution and concentrations in all genotypes used (Figure 5). This element was mainly located in the embryo and the aleurone layer (at concentrations between 1 and 2%) and was not detected in the endosperm. Sulfur was present in the whole embryo and the endosperm, and it was mainly located in the leaf and root primordia and the aleurone layer (at concentrations of approximately 0.2%), without any consistent difference among genotypes (Figure 5). It is also worth to remark the gradient of S concentrations, from high in the aleurone layer to low in the endosperm. ### DISCUSSION Enhancing NA synthesis via genetic transformation has been shown to increase the concentrations of Fe and Zn in rice seeds (Nozoye, 2018). However, it was still not known whether this was due to the enrichment in NA itself or to the subsequent enrichment in its derivative DMA, and whether the changes in the relative levels of both ligands may have an effect on the partitioning of metals between the embryo and endosperm. In this study we analyzed the concentrations of NA, DMA and metals in the embryo and endosperm of WT rice and six transgenic lines, overexpressing OsNAS1 and/or expressing barley NAAT (HvNAATb), which provided contrasting levels of DMA and NA. This allows for outlining three different DMA/NA scenarios for metal distribution in the rice seed (Figure 7) that are discussed below. Results show that increasing DMA alone led to Fe enrichment in the embryo and endosperm, whereas increasing DMA in combination with NA led to Fe and Zn enrichment in both tissues (Figure 7). ### First DMA/NA Scenario In a first scenario, an enhanced NA level would not be fully depleted because of the limited capacity to use NA for DMA synthesis (Figure 7). Lines complying with this scenario were those having an enhanced expression of OsNAS1 alone (N1 and N2), or in combination with a low expression of barley NAATb (ND1), and showed consistent enrichments in NA, DMA, Fe and Zn in the endosperm, and also to enrichments of NA, DMA and Fe in the embryo (Figures 1–3). In the endosperm of these lines, the increases in DMA and NA concentrations (6- to 10-fold and 6- to 29-fold, respectively) were much larger than those found for Fe and Zn (1.7- to 2.0-fold and 1.7- to 2.5-fold, respectively), and occasionally accompanied of moderate changes in other metals (e.g., a 1.4-fold increase for Cu in line ND1). Other rice transgenics complying with this DMA/NA scenario are those overexpressing OsNAS1-3 (Lee et al., 2009b) or HvNAS1 alone (Masuda et al., 2009), as well as OsNAS2 in combination with SoyferH1 (Trijatmiko et al., 2016), which showed concentration increases in polished seeds (endosperm) of 2- to 33-fold for DMA, 5- to 32-fold for NA, 2.0- to 7.5-fold for Fe and 2.2- to 3.8-fold for Zn. The same scenario occurs when overexpressing OsNAS1 in combination with HvNAATb (Banakar et al., 2017b), leading to increases in polished seeds of 33-, 160-, 4.0-, and 4.1 fold for DMA, NA, Fe, and Zn concentrations, respectively; that study also shows an increased abundance of NA and DMA in leaves and roots, which promotes Fe and Zn uptake, root-toshoot translocation, and finally seed loading. Other transgenics also showed concomitant Fe and Zn concentration increases, but the DMA and/or NA concentrations were not determined (Wirth et al., 2009; Boonyaves et al., 2017; Wu et al., 2018). The elemental images of these transgenic lines (N1, N2, and ND1) confirmed that embryos were enriched in Fe, and also showed changes in the metal distribution pattern, with increases in Fe concentrations in the leaf primordium, scutellum and root primordium (Figures 5, 6). The mobilization of Fe and Zn in the rice seed involves transport from the endosperm near the embryo to the epithelium, scutellum and then to the leaf and root primordia (Takahashi et al., 2009). In N1 and N2 embryos, the increases in NA and DMA concentrations (Figure 2) and low DMA/NA ratios (Supplementary Figure S2) would allow for the formation of Fe(II)-NA in addition to Fe(III)-DMA, with Fe transport occurring via YSLs such as OsYSL9/OsYSL2. OsYSL9 works with Fe(II)-NA and Fe(III)-DMA and is expressed in the endosperm adjacent to the embryo and scutellum (Senoura et al., 2017), whereas OsYSL2 functions with Fe(II)-NA [not with Fe(III)-DMA] and is expressed during seed development in the whole embryo (Koike et al., 2004) and in mature seeds in the epithelium, vascular bundle of the scutellum and leaf primordium (Nozoye et al., 2007). In contrast, the embryos of ND1 were also enriched in DMA and NA (Figure 2) but had a slight increase in the DMA/NA ratio (Supplementary Figure S2). These embryos also showed an accumulation of Fe in the epithelium, scutellum and root primordium, but Fe in the leaf primordium was reduced (Figures 5, 6). Since in this genotype Fe(III)-DMA would be favored over Fe(II)-NA, this reduction suggests that Fe transport to the leaf primordium occurs as Fe(II)-NA via OsYSL2, a transporter specific for Fe (and Mn) complexes with NA (but not with DMA) localized in the embryo (Koike et al., 2004). The Zn distribution pattern in the embryo was also altered in this scenario when compared to the WT (Figures 5, 6), especially in the leaf primordium, the structure with the highest Zn concentration. In the N1 and ND1 embryos, the extremely high levels of DMA + NA (6- and 4-fold higher than in the WT, respectively) resulted in Zn depletion not only of the leaf primordium, but also the root primordium when NA was as abundant as DMA (in N1) (Figures 5, 6). In contrast, the slight increase of DMA+NA in N2 (1.4-fold) resulted in smaller decreases in the Zn concentrations in the leaf primordium and increased Zn concentrations in the scutellum and root primordium. A large abundance of Zn chelators would diminish the pool of free Zn(II) ions, therefore limiting its availability for transport via OsZIP4 and/or OsIRT1 throughout the embryo, and more specifically toward the meristematic tissues where this metal tends to accumulate massively. OsZIP4 is expressed in the vascular bundle of the scutellum and the leaf and root primordium (Takahashi et al., 2009, 2011), whereas OsIRT1, which transports Zn in addition to Fe (Lee and An, 2009), is also expressed in embryo structures (Nozoye et al., 2007). ### Second DMA/NA Scenario In a second DMA/NA scenario, an enhanced NA level would be depleted by an enhanced capacity to use NA for DMA synthesis (Figure 7). The line complying with this scenario, ND2, had an enhanced expression of OsNAS1 in combination with a high expression of HvNAATb, and showed enrichments only for DMA and Fe, both in the endosperm and embryo, whereas NA and Zn concentrations in the embryo decreased (by 76 and 40%, respectively) and Cu concentrations increased (2.0-fold), without any change in the endosperm concentrations of NA, Zn, Mn and Cu (Figures 1–3). In this line, the endosperm enrichments for DMA and Fe (19- and 2.9-fold, respectively) were much larger than those found in the lines of the first scenario (see above). This scenario also occurs with the constitutive expression of the strict Fe(III)-DMA transporter HvYS1 (Murata et al., 2006), which leads to increases in the concentrations of both DMA (2.3-fold) and Fe (2.1-fold) in polished seeds, without affecting the concentrations of NA, Zn and Mn (Banakar et al., 2017a). In a fully opposite DMA/NA scenario, the Osnaat1 mutant shows a large increase in NA accompanied by a large DMA depletion, resulting in an stimulated Fe(II) acquisition and a seed enrichment in Fe (1.8- and 3.8-fold increases in unpolished and polished seeds, respectively) but not Zn (Cheng et al., 2007). The elemental images of ND2 seeds confirms that embryos were markedly enriched in Fe, and also showed changes in the metal distribution pattern, with an accumulation of Fe in the epithelium, scutellum and root primordium, and a depletion of Fe in most of the leaf primordium (Figure 5). This supports that the transport of Fe from the endosperm near the embryo to the epithelium, scutellum and root primordium can be via OsYSL9, mediated by Fe(III)-DMA in addition to Fe(II)-NA, since the extremely high levels of DMA and major depletion of NA (Figure 2) would strongly favor the formation of Fe(III)- DMA over Fe(II)-NA. On the other hand, the ND2 data also provide further support to the idea that the transport of Fe from the scutellum to the leaf primordium occur as Fe(II)-NA via OsYSL2. The decrease in Zn and increase in Cu in the embryo in ND2 provides some hints on the partitioning of both metals in the rice seed. The possible Zn transport forms within the rice grain include free Zn(II) ions, Zn(II)-NA, and Zn(II)- DMA, with the latter being unlikely to be relevant, since the Osnaat1 mutant shows no Zn partitioning phenotype in the grain (Cheng et al., 2007). In a previous study, the activation of OsNAS2 generated a new pool of bio-available Zn in the endosperm, mainly composed of Zn(II)-NA and Zn(II)-DMA (Lee et al., 2011). The excess of DMA in the embryo of ND2 may favor the formation of Zn(II)-DMA, hampering the transport of free Zn(II) ions from the endosperm via the highly selective transporter OsZIP4 (Ishimaru et al., 2005), expressed in the endosperm region adjacent to the epithelium in mature rice seeds (Takahashi et al., 2009). On the other hand, the excess of DMA and the depletion of NA in ND2 will difficult the formation of Zn(II)-NA, hampering transfer via YSLs using this metal chelate. OsYSL15 and OsYSL9 have not been assayed yet in this respect (Inoue et al., 2009; Lee et al., 2009a; Senoura et al., 2017), whereas others, including OsYSL2 (Koike et al., 2004), OsYSL16 (Zheng et al., 2012) and OsYSL18 (Aoyama et al., 2009) do not transport Zn(II)-NA. The Zn distribution pattern in the embryo was also altered in this scenario when compared to the WT (Figure 5), mainly affecting the leaf primordium, the structure with the highest Zn concentration. In the ND2 embryos, the extremely high levels of Zn chelators (DMA + NA) (10-fold higher than in the WT) resulted in a Zn depletion in the leaf primordium (Figure 5). As indicated above for N1 and ND1 (first DMA/NA scenario), the abundance of Zn chelators would tend to decrease the pool of free Zn(II) ions, therefore limiting transport via OsZIP4 and/or OsIRT1. Copper in the embryo was increased in ND2, conversely to what occurs with Zn. In this second scenario, which includes high DMA and low NA availability, Cu complexation is favored, since the stability constants are higher for Cu [18.7 for Cu(II)- DMA, Murakami et al., 1989; 18.6 for Cu(II)-NA, Beneš et al., 1983] than for Zn [12.7 for Zn(II)-DMA, Murakami et al., 1989; 15.4 for Zn(II)-NA, Anderegg and Ripperger, 1989]. A likely candidate for Cu delivery to the embryo is OsYSL16, which is highly expressed in all tissues of developing seeds (Lee et al., 2012), and transports Cu(II)-NA and Fe(III)-DMA, but not Cu(II)-DMA, Fe(II)-NA and Zn(II)-NA (Kakei et al., 2012; Zheng et al., 2012). It is also possible that YSL2, YSL9 and YSL18, which are expressed in embryo and/or endosperm during seed development (Koike et al., 2004; Aoyama et al., 2009; Senoura et al., 2017), could be responsible for Cu delivery to the embryo, since YSLs can transport a broad range of substrates [for instance, ZmYS1 transports Fe(III)-DMA, Zn(II)-DMA, Cu(II)- DMA, Fe(II)-NA, Ni(II)-NA and others; Schaaf et al., 2004; Murata et al., 2006]. ### Third DMA/NA Scenario In the third DMA/NA scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (Figure 7). Lines complying with this scenario were those expressing HvNAATb alone (D1 and D2), and resulted in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm (Figures 1–3). A low expression of HvNAATb (D1) led to moderate decreases in DMA (26%) and Fe (35%) in the endosperm and to an accumulation of Fe in the aleurone layer (Figure 5), whereas in the embryo NA and Fe also decreased moderately (17 and 24%, respectively, in both cases significantly at P ≤ 0.10), DMA increased (3.9 fold) and Fe was depleted in all embryo structures (Figure 5). The presence of low levels of DMA in the endosperm would make more difficult to compete with phytic acid present in the aleurone layer, since Fe(III)-DMA and Fe(III)-phytic acid have similar stability constants (18.4 and 18.2, respectively; Murakami et al., 1989; Torres et al., 2005), and consequently, Fe would stay in the aleurone layer, limiting its transport to the inner endosperm and subsequently to the embryo. The high HvNAATb expression in D2 caused moderate increases in DMA (1.9-fold) in the endosperm and moderate decreases in NA and Fe in the embryo, with the Fe distribution pattern being unaffected. ### CONCLUSION When the transgenic approach results in increases in the DMA concentration alone or in combination with NA (second and first DMA/NA scenarios, respectively), the prevalent mechanisms appear to be those based on Fe(III)-DMA, which enhance Fe transport and storage in the endosperm, likely using YSL transporters. When increases in DMA occur in combination with NA increases (first DMA/NA scenario), an additional mechanism based on Zn(II)-NA appears to be elicited, which boosts Zn transport and storage in the endosperm. However, when the transgenic approach results only in minor changes in the DMA levels (third DMA/NA scenario) there are no effects on the metal status in the seed. This knowledge can help designing future strategies for biofortification strategies in rice, using the selectivity of the different ligands and transporters. It should be kept in mind that in high-NA/DMA grains the bioavailability of Fe for mammals and humans is improved even when the Fe concentrations are unchanged (Zheng et al., 2010; Eagling et al., 2014). Our study demonstrates that a better understanding of transgenic plant phenotypes, using in-depth localized quantification of the targeted nutrients and related metabolites in plant tissues, will facilitate the application of more refined strategies for biofortification of staple crops. ### AUTHOR CONTRIBUTIONS PC, BF, and AÁ-F conceived and designed the experiments. RB obtained the plant material. PD-B performed the HPLC-ESI-MS(TOF) analysis, obtained the seed sections, and performed Perl's staining. SR-M and BF performed the LA-ICP-MS analysis. PD-B prepared and analyzed the results, and drafted the manuscript. TC, RP, and RB analyzed critically the results. AÁ-F, JA, BF, and PC wrote, reviewed, and edited the paper. All the authors read and approved the final manuscript. ### FUNDING This work was supported by the grants of the Spanish Ministry of Science, Innovation and Universities (AGL2016-75226-R, BIO2014-54426-P and AGL2017-85377-R, all co-financed with FEDER), Aragón Government (Group A09\_17R) and Generalitat de Catalunya (Grant 2017 SGR 828). PD-B was supported by a MINECO-FPI contract. RB was supported by a Ph.D. fellowship from the University of Lleida. SR-M was supported by a research contract from the Fundación Universidad de Oviedo (FUO-069-17). BF was supported by a MINECO research contract (RYC-2014-14985; "Ramón y Cajal Program"). ### REFERENCES ### ACKNOWLEDGMENTS The authors acknowledge J. J. Rios (CEBAS-CSIC, Murcia, Spain) for providing technical advice on Perl's staining. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01190/ full#supplementary-material in monocotyledonous plants. Transgenic Res. 5, 213–218. doi: 10.1007/ BF01969712 reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One 6:e24476. doi: 10.1371/journal.pone.0024476 fluorescence imaging of Fe, Zn, Mn, and Cu. Plant Soil 325, 39–51. doi: 10.1007/ s11104-009-0045-7 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Díaz-Benito, Banakar, Rodríguez-Menéndez, Capell, Pereiro, Christou, Abadía, Fernández and Álvarez-Fernández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Overexpression of a High-Affinity Nitrate Transporter OsNRT2.1 Increases Yield and Manganese Accumulation in Rice Under Alternating Wet and Dry Condition Bingbing Luo1,2† , Jingguang Chen2,3† , Longlong Zhu1,2, Shuhua Liu1,2, Bin Li1,2 , Hong Lu1,2, Guoyou Ye<sup>3</sup> , Guohua Xu1,2 and Xiaorong Fan1,2 \* <sup>1</sup> State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing, China, <sup>2</sup> Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China, <sup>3</sup> CAAS-IRRI Joint Laboratory for Genomics-Assisted Germplasm Enhancement, Agricultural Genomics Institute in Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China ### Edited by: Huixia Shou, Zhejiang University, China ### Reviewed by: Yuanhu Xuan, Shenyang Agricultural University, China Dong Liu, Tsinghua University, China Chuang Wang, Huazhong Agricultural University, China > \Correspondence: Xiaorong Fan [email protected] †These authors have contributed equally to this work #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 14 December 2017 Accepted: 25 July 2018 Published: 15 August 2018 ### Citation: Luo B, Chen J, Zhu L, Liu S, Li B, Lu H, Ye G, Xu G and Fan X (2018) Overexpression of a High-Affinity Nitrate Transporter OsNRT2.1 Increases Yield and Manganese Accumulation in Rice Under Alternating Wet and Dry Condition. Front. Plant Sci. 9:1192. doi: 10.3389/fpls.2018.01192 Nitrate and manganese (Mn) are necessary elements for the growth and development of rice in paddy soil. Under physiological conditions, we previously reported that the uptake of Mn in roots can be improved by the addition of external nitrate but not ammonium. To investigate the mechanism(s) of this phenotype, we produced plant lines overexpressing OsNRT2.1 and assessed Mn uptake under alternating wet and dry (AWD) and waterlogged (WL) conditions. Under AWD condition, we observed a 31% reduction in grain yields of wild type (WT) plants compared to WL condition. Interestingly, the overexpression of OsNRT2.1 could recover this loss, as OsNRT2.1 transgenic lines displayed higher grain yields than WT plants. We also observed 60% higher grain Mn in the transgenic lines in AWD condition and approximately 30% higher Mn in the grain of transgenic lines in WL condition. We further found that the overexpression of OsNRT2.1 did not alter Mg and Fe in the seeds in either growth condition. The reasons for the increased Mn content in OsNRT2.1 transgenic seeds in AWD condition could be explained by the elevated expression of OsNRAMP family genes including OsNRAMP3, OsNRAMP5, and OsNRAMP6 in node I, the panicleneck, and the flag leaves. The mechanism(s) underpinning the upregulation of these genes requires further investigation. Taken together, our results provide a new function of OsNRT2.1 in improving rice yields and grain Mn accumulation during water-saving cultivation patterns. This represents a new strategy for maintaining yield and improving food quality in a sustainable agricultural system. Keywords: rice, OsNRT2.1, manganese uptake, yield, nitrate ### INTRODUCTION Trace elements play a vital role in plant growth and development (Yan et al., 2006). All organisms require trace levels of manganese (Mn) for survival due to its necessity during plant metabolism and its participation in several important pathways (Socha and Guerinot, 2014) including the oxygen-evolving complex (OEX) of photosystem II (PS II). In addition, Mn plays an important 65* role during phosphoenolpyruvate carboxykinase activation and liquid metabolism (Dziwornu et al., 2018). Thus, it is required for photosynthesis indirectly by repressing thylakoid synthesis. In addition, manganese superoxide dismutase (MnSOD) is the major mitochondrial antioxidant defense enzyme (Shen, 2015) and Mn is a co-factor/activator of many enzymes involved in the catalysis of oxidation reduction, decarboxylation and hydrolytic reactions (Marschner, 1995; Xu et al., 2007). Mn deficiency is a global problem in agriculture (Hebbern et al., 2005). Mn deficient plants are more vulnerable to cold stress and infections by pathogens, leading to decreased crop yields (Marschner, 1995; Hebbern et al., 2005). Addressing this issue is problematic as Mn2<sup>+</sup> rapidly oxidizes when supplemented into fertilizers. In this regard, further knowledge of molecular mechanisms that can enhance Mn delivery are required. Several Mn transporters contribute to the uptake, transport and maintenance of Mn homeostasis in plants. The NRAMP family was shown to participate in Mn transport during early plant discoveries. In Arabidopsis, AtNRAMP1 localizes to the plasma membrane and displays root-specific expression where its function is to coordinate the absorption of Mn from soil (Cailliatte et al., 2010). OsNRAMP5 is mainly involved in Mn uptake and accumulation in rice and its silencing significantly reduces Mn accumulation in shoots (Yang et al., 2014). OsNRAMP3 is expressed in the node and regulates Mn transport and tissue distribution in response to environmental changes (Yamaji et al., 2013a). OsNRAMP6 distributes to the plasma membrane and transports Mn and Fe, maintaining their balance in cells (Peris-Peris et al., 2017). In rice, Mn homeostasis is controlled by the YSL2/6 gene. OsYSL2 can promote the long-distance transport of Mn (Koike et al., 2004; Ishimaru et al., 2010). OsYSL6 belongs to the Mn-nicotianamine (NA) transporter family and is required for the detoxification of high concentrations of Mn (Sasaki et al., 2011). In addition, the CAX proteins belong to the Ca2+/cation antiporter (CaCA) superfamily (Emery et al., 2012) and are potentially involved in Mn2+/H<sup>+</sup> exchange to export Mn from the cytosol (Connorton et al., 2012). Nitrogen (N) is an essential element for plant growth and development, especially for crops. Generally, N is absorbed by plants in the form of ammonium (NH<sup>4</sup> <sup>+</sup>) and nitrate (NO<sup>3</sup> <sup>−</sup>), but nitrate easily dissolves in water and is therefore lost to the environment (Jin et al., 2015). Roots acquire NO<sup>3</sup> − via transporters distributed throughout the whole plant (Xu et al., 2012). Plants adapt to the differing NO<sup>3</sup> <sup>−</sup> concentrations in soil by exploiting two forms of NO<sup>3</sup> <sup>−</sup> uptake, including low-affinity transporters (NRT1/NPF) and high-affinity NO<sup>3</sup> − transporters (NRT2) (Crawford and Glass, 1998). Particularly for rice plants, we previously identified a high-affinity NO<sup>3</sup> − transport system. The OsNRT2 gene family was found to play an important role during N uptake and translocation, requiring their partner protein NAR2 to perform this function, besides OsNRT2.3b (Tang et al., 2012; Xu et al., 2012; Chen et al., 2016b; Chen Z.C. et al., 2017; Fan et al., 2016; Chen J.G. et al., 2017). Simultaneously, Mn can influence NO<sup>3</sup> <sup>−</sup> reductase activity and is associated with photosynthesis in plants (Botrill et al., 1970; Gong et al., 2011). Mn also influences N metabolism and regulates protein synthesis (Jiang, 2006). Studies have shown that the arabidopsis chl1-5 mutant lines display reduced NO<sup>3</sup> <sup>−</sup> uptake and a loss of AtIRT1 expression, which is responsible for Cd uptake into root cells (Muños et al., 2004; Lux et al., 2011). Fe deficiency was also shown to inhibit N metabolism in the roots and leaves of cucumber plants (Borlotti et al., 2012). These effects suggest that NO<sup>3</sup> <sup>−</sup> influences the uptake of trace elements in plants. In this study, we hypothesized that a close relationship between N and Mn in plants exists. We used transgenic rice overexpressing OsNRT2.1 to examine how the different forms of N influence Mn uptake and accumulation in grain. ### MATERIALS AND METHODS ### Plant Materials and Growth Conditions We amplified the OsNRT2.1 (AB008519) ORF (primers are displayed in Supplementary Table S1) using cDNA obtained from Oryza sativa L. ssp. Japonica cv. Nipponbare. PCR products were cloned into the pMD19-T vector (TaKaRa Biotechnology, Dalian, China) and the expression vector pTCK303 containing a ubiquitin promoter. Positive clones were verified by restriction digest analysis and DNA sequencing. Next, the binary vector pUbiquitin-OsNRT2.1 was introduced into A. tumefaciens (strain EHA105), which was used to transform the rice embryonic callus as previously described (Ai et al., 2009). Hygromycin-resistant T0 generation transgenic rice plants were transplanted to soil and grown to obtain seeds in fields (Tang et al., 2012). Three independent T4 generation lines overexpressing OsNRT2.1 were used for further experiments. Firstly, rice seedlings were selected and cultured in 1 mM (NH4)2SO<sup>4</sup> as the main source of N in nutrient solution (pH 5.5) for 1 month. Other elements and trace elements were supplied in IRRI (International Rice Research Institute) nutrient solution containing 0.35 mM K2SO4, 0.3 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 0.5 mM Na2SiO3, 20 µM H3BO3, 9 µM MnCl2, 20 µM EDTA-Fe, 0.77 µM ZnSO4, 0.32 µM CuSO4, and 0.39 µM (NH4)6Mo7O24. Rice were planted in a growth room (Thermoline Scientific Equipment Pty. Ltd., Smithfield, NSW, Australia) at 30◦C during the day and 22◦C at night with 16-h light/8-h of darkness. The light intensity was 400 µmol m−<sup>2</sup> s −1 and the relative humidity was 65– 70%. Wild type (WT) rice were then transferred to 0.25 or 1.25 mmol/L Ca(NO3)<sup>2</sup> and 0.25 or 1.25 mmol/L (NH4)2SO<sup>4</sup> nutrient solution, respectively, for 2 weeks (Figure 1). In Figures 2 and 4, WT and overexpression lines were transferred to 0.5 mM NH<sup>4</sup> <sup>+</sup>/NO<sup>3</sup> <sup>−</sup> nutrient solution for 2 weeks. For each line and treatment, four biological repeats were performed. In field experiments, rice were planted in Nanjing, Jiangsu; a subtropical monsoon climate zone. The characteristics of the soil and N supply were as previously described (Chen et al., 2016b). For waterlogged (WL) treatment, rice fields were watered daily to maintain WT and transgenic lines in a flooded state. For alternating wet and dry treatment (AWD), lines were planted into fields and watered for a week, to keep the soil moist. ### Southern Blot Analysis Transgene cope numbers were identified by southern blot analysis. Briefly, genomic DNA was extracted from the leaves of WT and transgenic lines and digested with HindIII and EcoRI. Digested DNA was separated on 1% (w/v) agarose gels, transferred to a Hybond-N<sup>+</sup> nylon membrane and hybridized using the hygromycin-resistant gene. ### RNA Extraction and qPCR Analysis Total RNA was extracted from 100 mg of tissue using TRIzol (Invitrogen, Carlsbad, CA, United States). Total RNA concentrations were assessed by UV spectrophotometry (Eppendorf, Bio-photometer, Germany). RNA (2 µg) was reverse transcribed into cDNA using HiScript Reverse Transcriptase (Vazyme, Nanjing, China) according to the manufacturers protocol. Four biological repeats were performed for each qPCR reaction, using OsActin as a reference gene. Primers were designed to detect OsNRT2.1, OsNRT2.3a, OsNRT2.4, OsNAR2.1, OsNRAMP3, OsNRAMP5, OsNRAMP6, OsIRT1, and OsMGT1 and are listed in Supplementary Table S2. PCR amplification was performed using SYBR qPCR Master Mix (Vazyme, Nanjing, China). PCR reactions were performed under the following parameters: 95◦C for 30 s, followed by 40 cycles of 95◦C for 10 s, 60◦C for 30 s, and 72◦C for 10 s. #### Determination of the <sup>15</sup>N-NH<sup>4</sup> <sup>+</sup>/NO<sup>3</sup> − Influx Rate in Different Rice Lines Rice seedlings of WT and OsNRT2.1 transgenic rice plants were planted in IRRI solution containing 1 mM NH<sup>4</sup> <sup>+</sup> for 2 weeks and N starved for 3 days. Plants were first transferred into 0.1 mM CaSO<sup>4</sup> for 1 min, then to complete nutrient solution containing either 0.5 mM <sup>15</sup>NH<sup>4</sup> <sup>+</sup> or 0.5 mM <sup>15</sup>NO<sup>3</sup> <sup>−</sup> (atom% <sup>15</sup>N: 99%) for 5 min and finally to 0.1 mM CaSO<sup>4</sup> for 1 min (Duan et al., 2007). The <sup>15</sup>N influx rate was calculated according to methods described by Tang et al. (2012). ### Assessment of Dry Weight, Total N and Metal Ion Accumulation To investigate the links between OsNRT2 function and metal ion uptake, we investigated the levels of metal elements using the ICP-OES method in OsNRT2.1 transgenic lines and Mn elements in OsNRT2.3a/b transgenic lines. The creation and identification processes of bO-1, bO-2, bO-8 for OsNRT2.3b transgenic lines and aO-1, aO-2 for OsNRT2.3a transgenic lines were performed as previously described (Fan et al., 2016). Fresh WT or transgenic lines were harvested at the rice mature stage (n = 4) and heated at 105◦C for 30 min. Panicles, flag leaves, second and third leaves, sheaths and stems were then dried for 3 days at 75◦C. Rice obtained from hydroponic experiments was divided into shoots and roots only. Dry weights were recorded as biomass values. Using the Kjeldahl method (Li et al., 2006), total N accumulation was assessed in the different plant areas through multiplying the N concentration by the corresponding biomass. Dried samples were wet-digested in concentrated HNO<sup>3</sup> at 120◦C until no brown nitrogen oxide gas was emitted. When the samples became transparent, they were further digested with HClO<sup>4</sup> at 180◦C. Samples were then diluted with ultrapure water and the concentrations of metal elements in the digestates were analyzed using ICP-OES (iCAP 6300). ### Statistical Analysis All data were analyzed using the Tukey's test of one-way analysis of variance (ANOVA). Statistically significant differences at the p < 0.05 level (one-way ANOVA) between transgenic and WT and/or between other treatments were assessed. All statistical evaluations were performed using IBM SPSS Statistics version 20 software (SPSS Inc., Chicago, IL, United States). ### RESULTS ### Assessment of Mn Absorption Under Different N Treatments Wild type rice seedlings were planted under different conditions of N supply. Symptomatically, the roots of rice seedlings were better in 0.5 mM NO<sup>3</sup> <sup>−</sup> than in 0.5 mM MH<sup>4</sup> +, 2.5 mM NO<sup>3</sup> <sup>−</sup> and 2.5 mM NH<sup>4</sup> <sup>+</sup> conditions (Figure 1A). Statistical analysis showed that the dry weights of the plant roots in 0.5 mM NO<sup>3</sup> <sup>−</sup> condition were significantly increased (Figure 1B). Low concentration NO<sup>3</sup> <sup>−</sup> could promote root elongation and increase root hairs (Kiba and Krapp, 2016). For the whole plant, the dry weight was best in 2.5 mM N, with no differences between 2.5 mM NO<sup>3</sup> <sup>−</sup> and 2.5 mM NH<sup>4</sup> <sup>+</sup> observed (Figure 1C). Next, the total N in rice seedlings was investigated. Total N content in rice seedlings with 2.5 mM N supply was higher than that of the 0.5 mM N supply (Supplementary Figure S1). Rice seedlings planted in 2.5 mM NH<sup>4</sup> <sup>+</sup> nutrient solution, displayed the best outcome (Supplementary Figure S1). Simultaneously, the Mn concentration and content of rice roots in 0.5 mM NO<sup>3</sup> <sup>−</sup> was found to increase more than other conditions. However, shoots were lowest in 2.5 mM NH<sup>4</sup> <sup>+</sup> solution (Figures 1D,E). The Mn content of rice seedlings in NO<sup>3</sup> <sup>−</sup> solution was higher than in NH<sup>4</sup> <sup>+</sup> using the same N concentrations (Figure 1F). In addition, the expression of the nitrate transporters OsNRT2.1/OsNRT2.3 were up-regulated by external NO<sup>3</sup> <sup>−</sup> and the expression of OsNAR2.1 increased in 0.5 mM NO<sup>3</sup> <sup>−</sup>/2.5 mM NO<sup>3</sup> − compared to NH<sup>4</sup> <sup>+</sup> treatments in the different tissues (Supplementary Figures S2A–C). The expression of Mn transporters OsNRAMP3/OsNRAMP5/OsNRAMP6 also increased following NO<sup>3</sup> <sup>−</sup> treatment compared with NH4<sup>+</sup> treatment. Taken together, these results reveal that both Mn uptake and OsNRAMP3/OsNRAMP5/OsNRAMP6 expression are increased by NO<sup>3</sup> <sup>−</sup>. Therefore, NO<sup>3</sup> <sup>−</sup> positively regulates the absorption of Mn in rice. ### Assessment of the Expression Patterns of OsNRT2s and OsNAR2.1 in the Roots of Transgenic Lines Firstly, transgenic lines were identified by southern blot analysis and RT-PCR. The data showed that three transgenic lines were one copy insertions and OsNRT2.1 was overexpressed to approximately five-fold higher mRNA levels in roots and shoots under normal N conditions (1.25 mM NH4NO<sup>3</sup> supply) (Supplementary Figure S4 and Chen et al., 2016a). WT and transgenic OsNRT2.1 lines were planted in 0.5 mM NO<sup>3</sup> <sup>−</sup>/NH<sup>4</sup> + nutrition solution, respectively. RT-PCR was performed to confirm the gene expression patterns of the two families of NO<sup>3</sup> <sup>−</sup> transporters in WT and OsNRT2.1 transgenic lines under different N supplies. OsActin was used as a reference gene for comparison. Total RNA was extracted from the rice roots of the different lines. Under conditions of low concentration (0.5 mM) of NH<sup>4</sup> <sup>+</sup> and NO<sup>3</sup> <sup>−</sup>, the expression of OsNRT2.1 in transgenic lines increased 4.5-fold and 5.7-fold, compared to WT (Figures 2A,B). No differences in the relative expression of other NO<sup>3</sup> <sup>−</sup> transporters OsNRT2.3a/OsNRT2.4 between transgenic and WT lines or between NO<sup>3</sup> <sup>−</sup> and NH<sup>4</sup> <sup>+</sup> treatments were observed (Figures 2A,B). However, the expression levels of OsNAR2.1 increased approximately 80% in transgenic lines in 0.5 mM NO<sup>3</sup> <sup>−</sup>, but not in 0.5 mM NH<sup>4</sup> <sup>+</sup> (Figures 2A,B). The total N content of the three transgenic lines was higher than WT in the roots and the shoots under 0.5 mM NO<sup>3</sup> <sup>−</sup> conditions, with no differences in the NH<sup>4</sup> <sup>+</sup> solution observed (Supplementary Figure S5). Taken together, these results show that OsNRT2.1 expression is enhanced in the transgenic rice. In addition, the expression of OsNRT2.1 and OsNAR2.1 is enhanced in all transgenic lines, allowing an efficient transfer of NO<sup>3</sup> <sup>−</sup> in 0.5 mM NO<sup>3</sup> − conditions. #### NH4<sup>+</sup> and NO<sup>3</sup> <sup>−</sup> Influx Rates in WT and OsNRT2.1 Transgenic To confirm the influence of OsNRT2.1 on high-affinity root NO<sup>3</sup> <sup>−</sup> influx into intact plants, short-term nitrate absorption was assessed by transferring all the lines to either 0.5 mM <sup>15</sup>NH<sup>4</sup> <sup>+</sup> or 0.5 mM <sup>15</sup>NO<sup>3</sup> <sup>−</sup> for 5 min. Under 0.5 mM <sup>15</sup>NH<sup>4</sup> <sup>+</sup> treatment condition, the three transgenic lines displayed no significant differences to WT (Figure 3A). However, OsNRT2.1 transgenic lines were enhanced by 19% compared to WT during NO<sup>3</sup> − influx (Figure 3B). In addition, the effects of overexpression on rice growth under different forms of N supply were studied by comparing the total N concentration and content in different parts of the rice plants. The total N of the transgenic lines did not significantly differ in the roots and shoots compared to WT lines in 0.5 mM NH<sup>4</sup> <sup>+</sup> solution (Supplementary Figures S5A,B). However, the total N content of the roots and shoots of the transgenic rice plants was enhanced by 97% and 36%, respectively, compared to WT lines in 0.5 mM NO<sup>3</sup> <sup>−</sup> conditions (Supplementary Figure S5E). Total N concentrations in the shoots did not differ from WT (Supplementary Figure S5D). These results show that the overexpression of the high-affinity nitrate transporter OsNRT2.1 improves NO<sup>3</sup> <sup>−</sup> uptake in 0.5 mM NO<sup>3</sup> <sup>−</sup>, compared to WT. ### Mn Concentration of Shoots and Roots of Transgenic Plants Under Different N Treatments The transferability of Mn is weak. From hydroponic experiments (Figure 4), we tested the Mn concentration of shoots and roots in rice when planted in different nutritive forms of N. We found that the dry weight of roots and shoots increased by 66 and 29%, respectively, in transgenic lines relative to WT lines in 0.5 mM NO<sup>3</sup> <sup>−</sup> solution (Figure 4B). However, dry weights did not significantly differ in 0.5 mM NH<sup>4</sup> <sup>+</sup> (Figure 4A). Simultaneously, Mn concentrations of roots and shoots in the overexpression lines were also enhanced by 43% and 47%, respectively, in 0.5 mM NO<sup>3</sup> <sup>−</sup> solution, but not in 0.5 mM NH<sup>4</sup> <sup>+</sup> (Figures 4C,D). From Figure 4 and Supplementary Figure S3, we reasoned that this was due to the OsNRT2.1 gene transferring NO<sup>3</sup> <sup>−</sup> into the rice, increasing total N, Mn uptake and accumulation in 0.5 mM NO<sup>3</sup> <sup>−</sup> condition. These results indicate that Mn assimilation by OsNRT2.1 is NO<sup>3</sup> − uptake dependent, and that the overexpression of OsNRT2.1 does not only increase NO<sup>3</sup> <sup>−</sup> uptake to enhance total N, but also promotes Mn absorption in rice in low NO<sup>3</sup> − condition. ### Effects of Different Irrigation Conditions on N and Mn Concentrations in Grain Rice typically grows in anaerobic flooded fields, which exist mainly in the form of NH<sup>4</sup> <sup>+</sup>-N. Conversely, NO<sup>3</sup> <sup>−</sup> is present mainly in aerobic uplands (Stitt, 1999). To simulate hydroponic conditions in the presence of different N treatments, we designed a field experiment under different irrigation conditions and investigated OsNRT2.1 function on rice grains in the field. From the assessment of seed morphology, seeds of WT under alternating wet and dry (AWD) condition were shorter than other seeds (Figure 5A and Supplementary Figure S6). Compared to other field treatments, we found that the grain weight of WT in AWD condition was approximately 31% lower than waterlogged (WL) condition, with no differences in the transgenic lines observed (Figure 5B). In addition, no evident differences in all lines in WL condition were observed. However, the grain weight of transgenic rice plants was approximately 26% higher compared to WT weights in AWD condition (Figure 5A). The 1000-grain weight displayed a similar pattern to the grain weights (Figure 5B). We also tested the total N concentration of the seeds under different field treatments. Interestingly, WT and transgenic lines were higher in AWD condition compared to waterlogged condition, and the total N concentration of the transgenic seeds also increased by 15% compared to WT in the AWD field (Figure 5C). However, the total N concentration of the husk in the overexpression lines was lower than that of WT in the AWD field, whilst no differences in all lines from the WL field were observed (Supplementary Figure S7A). As higher levels of N were transferred into the seeds of transgenic lines in the AWD field, their seed weights were higher than WT. Simultaneously, the Mn concentrations in the seeds of transgenic lines in AWD condition were enhanced when compared to WL condition. No differences were observed for the different field conditions in WT lines (Figure 5D). In addition, the husk of grain displayed similar results in terms of Mn concentrations (Supplementary Figure S7B). The concentration of Fe and Mg in seeds and husk appeared to vary irregularly (Supplementary Figure S7). This presented the unity of the Mn element. These results demonstrate that rice planted in AWD condition displays higher total N and Mn concentrations in grain, particularly for OsNRT2.1 transgenic lines. We extracted total RNA from the culm of all lines planted in the two types of irrigated field. From Figures 5F,G, the relative expression of OsNRT2.1 in transgenic lines was higher than WT lines in WL and AWD conditions. However, OsNAR2.1 expression was enhanced 2.8-fold only in AWD field relative to WT. Therefore, the soil of AWD primarily existed in NO<sup>3</sup> <sup>−</sup> form to enhance NO<sup>3</sup> <sup>−</sup> uptake through increased OsNRT2.1 expression, leading to the induction of OsNAR2.1 expression. As the relative expression of OsNRT2.1 and OsNAR2.1 increase following AWD treatment, NO<sup>3</sup> <sup>−</sup> uptake may further improve Mn uptake compared to the WL field. ### Assessment of the Expression of Related Genes, Total N and Mn Accumulation During Maturity Stages in AWD Conditions To understand mechanism(s) of how OsNRT2.1 improves total N and Mn accumulation at the mature stage in AWD field, we extracted total RNA from the different areas of rice (Supplementary Figure S9) and assessed the expression of OsNRT2.1, OsNAR2.1, and Mn transporters-OsNRAMP3, OsNRAMP5, and OsNRAMP6 (Yamaji et al., 2013a; Yang et al., 2014; Peris-Peris et al., 2017). From Figure 6, the expression of the related nitrate genes-OsNRT2.1 and OsNAR2.1 in the three transgenic lines were higher in the panicle-neck, flag leaves, flag leaves sheaths and node I compared to WT rice. The panicle-neck connects vegetative and reproductive organs. Flag leaves are functional leaves for transferring nutrients. Studies have reported that high-affinity nitrate OsNRT2.1 requires its partner protein OsNAR2.1 to transfer nitrate (Feng et al., 2011; Yan et al., 2011; Tang et al., 2012). Accordingly, the expression of OsNRT2.1 and its partner protein OsNAR2.1 increased in the Panicle-neck and in the functional leaves at maturity. NO<sup>3</sup> <sup>−</sup> was transferred to the panicle to enhance total N accumulation in seeds, and further improve grain yields. Interestingly, we found that the expression of the Mn transporters: OsNRAMP3, OsNRAMP5, and OsNRAMP6 were also upregulated in transgenic lines. These genes displayed similar expression patterns to NO<sup>3</sup> <sup>−</sup> transporters (Figures 6E–G). In particular, the expression of OsNRAMP3 and OsNRAMP6 in the transgenic lines increased by 87 and 311% in comparison to WT in the node I, respectively (Figure 6H). Node I represents the junction of the vascular system connecting the leaves, stems and panicles. Therefore, Mn transporter genes-OsNRAMP3 and OsNRAMP6 preferentially transport Mn to flag leaves and the panicle during the late stages of plant growth in rice. We found that the biomass of transgenic and WT lines displayed no significant differences at maturity (Supplementary Figure S10A). The NO<sup>3</sup> <sup>−</sup> concentrations of the different plant areas (except for leaves in the overexpression lines) were higher than WT (Supplementary Figure S10D). However, total N accumulation did not differ in various parts of the plants, and Mn showed an irregular trend without flag leaves (Supplementary Figure S10). These results suggest that total N and Mn are transferred to grains from vegetative organs at maturity. We further assessed Fe and Mg content in various parts of the different lines, in which we observed no differences (Supplementary Figure S11). When the relative expression of OsIRT1 and OsMGT1 that represent Fe and Mg related genes (Lee and An, 2009; Chen Z.C. et al., 2017) were analyzed, the expression patterns were also inconsistent in diverse areas of the transgenic rice plants (Figure 7). Taken together, these data suggest that the improvement of Mn concentration in OsNRT2.1 lines was due to the increased expression of Mn transporters, but no effects on other metal elements were observed. ## DISCUSSION Nitrate and Mn are essential nutrients in plants, and it has been reported that Mn deficiency decreases N uptake and metabolism (Gong et al., 2011). Excessive NO<sup>3</sup> <sup>−</sup> was shown to enhance Cd uptake in Thlaspi caerulescens (Xie et al., 2009) and wheat (Li et al., 2011). In addition, crosstalk between mineral elements exists. Wang et al. (2015) reported that Al improves Mn uptake and accumulation in rice roots, However, these tactics do not enhance the security of crops for human consumption because they do not increase the accumulation of beneficial elements in plant. In this study, the main objective was to investigate how interactions between Mn and NO<sup>3</sup> <sup>−</sup> influence rice growth and nutrient accumulation in roots, leaves tissues and grain. We found that NO<sup>3</sup> <sup>−</sup> improves Mn uptake in rice (Figure 2). When Mn concentrations were assessed in OsNRT2.1/OsNRT2.3a/b transgenic lines planted in normal field which was WL condition, respectively (Supplementary Figure S3), we observed increased Mn in the seeds and husk of OsNRT2.1 (Supplementary Figures S3A,D), but no enhanced uptake in OsNRT2.3a/b transgenic lines (Supplementary Figures S3B,C,E,F). Given these data, we investigated the pattern of Mn accumulation in OsNRT2.1 transgenic lines in further detail, under differing conditions of N supply and field FIGURE 6 \| Relative expression of related genes in different arears of WT and transgenic plants in AWD fields. Total RNA was isolated from (A,E) panicle-neck, (B,F) flag leaves, (C,G) flag leaves sheaths, and (D,H) Node I of WT and transgenic lines. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between WT and overexpression lines (P < 0.05, one-way ANOVA). conditions. This information is important as rice typically grows in anaerobic flooded fields, in which N exists mainly in the form of NH<sup>4</sup> <sup>+</sup>, as opposed to aerobic uplands where the major form of N is NO<sup>3</sup> <sup>−</sup> (Stitt, 1999). We found that OsNRT2.1-regulates NO<sup>3</sup> <sup>−</sup> uptake in roots, which in turn increases Mn root entry. This increases the Mn concentration in rice grain in the presence of low concentrations of NO<sup>3</sup> <sup>−</sup> and under AWD condition. Thus, enhancing NRT2.1-mediated NO<sup>3</sup> <sup>−</sup> uptake represents an attractive mechanism of increasing Mn accumulation in food. #### Effects of NO<sup>3</sup> <sup>−</sup> and NH<sup>4</sup> <sup>+</sup> Nutrition on Mn Accumulation We demonstrate that NO<sup>3</sup> <sup>−</sup> nutrition promotes Mn assimilation in plants to higher levels than NH<sup>4</sup> <sup>+</sup> nutrition (Figure 1). Thus Mn availability in nutrient solutions is influenced by the type of N-nutrient treatments. In 0.5 mM NH<sup>4</sup> <sup>+</sup>, Mn uptake in the roots did not differ in OsNRT2.1 transgenic lines (Figure 4C). However, Mn uptake drastically increased in the transgenic lines in 0.5 mM NO<sup>3</sup> <sup>−</sup> (Supplementary Figure S5 and Figure 4D). It was recently shown that NO<sup>3</sup> <sup>−</sup> uptake induces external alkalization, reducing Fe/Mn concentrations by enhancing the levels of H2O<sup>2</sup> in rice (Chen et al., 2018). In this study, we performed hydroponic experiments in MES buffered nutrient medium (to control pH) and nutrient treatments were replaced every 2 days. Furthermore, the Mn content in grain from field experiments should not be influenced by pH as the rhizosphere ranges from pH 5.5 to 6.0 in paddy soil (Pan et al., 2016). Thus, any effects of soil alkalization were excluded. We thus hypothesize that NO<sup>3</sup> <sup>−</sup> upregulates the expression of Mn transporters, including OsNRAMP3, OsNRAMP5, and OsNRAMP6 to increase Mn uptake and accumulation (Figure 1 and Supplementary Figure S2). We verified the expression of OsNRT2.1 and OsNAR2.1 under NH<sup>4</sup> + and NO<sup>3</sup> <sup>−</sup> conditions in transgenic OsNRT2.1 lines and found that OsNRT2.1 was unaffected by the different N forms. However, the expression of its partner protein OsNAR2.1 was significantly up-regulated in 0.5 mM NO<sup>3</sup> <sup>−</sup> (Figure 2). The OsNRT2.1 lines could still promote NO<sup>3</sup> <sup>−</sup> uptake (Figure 3) into the different tissues compared to WT plants (Supplementary Figure S10D). Thus, the up-regulation of OsNAR2.1 expression in NO<sup>3</sup> − condition (Figure 2B) promotes NO<sup>3</sup> <sup>−</sup> uptake in transgenic plants compared with WT (Supplementary Figure S10D). The observation that the upregulation of OsNRT2.1/OsNAR2.1 is favorable to the transport of NO<sup>3</sup> <sup>−</sup> in plants and improves rice yield, is consistent with our previous findings (Chen et al., 2016b; Chen J.G. et al., 2017). As we did not observe enhanced expression of either OsNRT2.3a or OsNRT2.4 lines in NO<sup>3</sup> <sup>−</sup> condition, we speculate that the regulation of OsNAR2.1 differs from other OsNRT2 genes according to the plant NO<sup>3</sup> − content (Supplementary Figure S2, Yan et al., 2011; Wei et al., 2018). ### AWD vs. WL Conditions in WT vs. Transgenic Lines In field experiments, the grain weight of WT lines under AWD condition decreased by 31% compared to WL condition. The NO<sup>3</sup> <sup>−</sup> concentrations in OsNRT2.1 transgenic lines also differed across plant areas in AWD condition and which were higher than WT lines (Supplementary Figure S10D). However, the total N concentration did not significantly differ across the lines (Supplementary Figure S10C). The total N of seeds in transgenic lines increased compared to WT (Figure 5). Thus, the overexpression of OsNRT2.1 improves NO<sup>3</sup> <sup>−</sup> uptake and assimilation efficiency to increase N accumulation in grain, leading to enhanced grain yields. In hydroponic experiments, the overexpression of OsNRT2.1 also enhanced NO<sup>3</sup> <sup>−</sup> uptake (Figure 3B) and N accumulation (Figure 5D), maintaining plant grain yields in WL condition. This is because in AWD condition, a high concentration of dissolved oxygen is present, which can influence nitrification by nitrifying bacterial, or chemical oxidation for the conversion of NH<sup>4</sup> <sup>+</sup> to NO<sup>3</sup> <sup>−</sup> at the root surface (Li et al., 2008; Steffens et al., 2011). Thus, under AWD condition, NO<sup>3</sup> <sup>−</sup> plays an important role in N accumulation and contributes to enhanced grain yields. However, for WT plants, the capacity to uptake NO<sup>3</sup> <sup>−</sup> is limited; and thus, grain yields are dramatically reduced. This observed loss of grain in WT type rice can likely be explained by a multitude of mechanisms. Surprisingly, we found that under AWD, Mn in the grain of transgenic plants was greatly increased compared to WL condition, but in WT rice, no changes were evident (Figure 5E). In addition, in WL condition, Mn levels also increased in the transgenic lines compared to WT (Figure 5E). We observed no differences in Mn concentrations in other parts of the plant under AWD condition (Supplementary Figure S10). Thus, higher levels of Mn were transported to grain and accumulated (Figure 5). This explains the improvement in seed quality, emergence, and seeding growth observed, as the positive effects of Mn on these processes is well documented (Dimkpa and Bindraban, 2016). The seeds of OsNRT2.1 overexpression lines not only increased in their total N accumulation, but enhanced Mn content was also observed (Figure 5). The length/width of these seeds were also better than WT (Figure 5A and Supplementary Figure S6), demonstrating that Mn plays an important role in increasing crop nutritional quality, crop yield and biomass production. Other metal elements such as Mg and Fe were not influenced by OsNRT2.1 overexpression (Supplementary Figures S8, S11). ### Enhanced Expression of Mn Transporters Explains Enhanced Mn Uptake in Transgenic Lines We verified gene expression profiles in the organs responsible for grain filling and discovered that the expression of OsNRT2.1 and OsNAR2.1 were enhanced in the panicle-neck, flag leaves and sheaths (Figure 6A). In the same plant areas, the expression of OsNRAMP5 and OsNRAMP6 increased in the OsNRT2.1 lines. Interestingly, OsNRAMP3/6 expression was enhanced in node I (Figure 6B). The expression of related genes involved in Mg and Fe uptake were also altered by OsNRT2.1 overexpression (Figure 7). It is understood that node I is a junction of vasculatures that link leaves, stems and panicles and so is important for the transport of nutrient elements into grain (Yamaji and Ma, 2009, 2014; Yamaji et al., 2013a). Transporters responsible for the delivery of minerals into seeds have been reported, including OsYSL16 for Cu (Zheng et al., 2012), OsHMA2 for Zn and Cd (Yamaji et al., 2013b) and AtNIP6;1 that is expressed in the node region for B distribution (Tanaka et al., 2008). Accordingly, the majority of these genes are also strongly expressed in node I (Tanaka et al., 2008; Yamaji and Ma, 2009, 2014; Zheng et al., 2012; Yamaji et al., 2013a). ### CONCLUSION Taken together, we show that AWD treatment can induce the expression of NO<sup>3</sup> <sup>−</sup> and Mn transporters in grain filling organs which increases the accumulation of N and Mn in grain. NO<sup>3</sup> <sup>−</sup> uptake in OsNRT2.1 transgenic lines can improve Mn accumulation, however, the Mn concentration does not increase in the seeds and husk of OsNRT2.3a/b overexpression lines, which also display increased NO<sup>3</sup> <sup>−</sup> uptake compared to WT lines (Fan et al., 2016). Thus, the mechanism(s) linking NO<sup>3</sup> <sup>−</sup> and Mn in OsNRT2.1 overexpressing plants differ from other OsNRT2 overexpression lines and is worthy of further investigation. From our findings, we propose a new application to improve both N and water efficiency in agricultural systems and demonstrate how high OsNRT2.1 expression improves Mn content in rice grain. ### AUTHOR CONTRIBUTIONS BiL, JC, and XF conceived the study, analyzed the data, and drafted the manuscript. BiL, LZ, and SL cultivated the rice materials and collected the rice samples. LZ, BL, and HL extracted RNA and performed the qRT-PCR experiments. BL and LZ participated in field and material management. BiL and JC conducted the statistical analysis of raw data. XF, GX, and GY revised the manuscript. All authors read and approved the final manuscript. ### FUNDING This study was financially supported by China National Key Program for Research and Development (2016YFD0100700), National Natural Science Foundation (Grant No. 31372122), Jiangsu Science Fund for Distinguished Young Scholars (Grant No. BK20160030), the Transgenic Project (Grant 2016ZX08001003-008). ### ACKNOWLEDGMENTS We are also grateful for the Anhui Provincial Natural Science Foundation of China Science Foundation of China (No. 1608085MC59) and Major Special Science and Technology Project of Anhui Province (No. 16030701102). ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01192/ full#supplementary-material FIGURE S1 \| Assessment of the total N-content under different N treatments. (A) total N concentration, (B) total N content of roots and shoots from different N treatments, (C) total N content of whole plants. 0.5/2.5A: 0.5 mM/2.5 mM NH<sup>4</sup> + as an N source; 0.5/2.5 N: 0.5/2.5 mM NO<sup>3</sup> <sup>−</sup> as an N source. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between N treatments (P < 0.05, one-way ANOVA). FIGURE S2 \| Expression pattern of NO<sup>3</sup> <sup>−</sup> transporters and Mn transporters under different N treatments; total RNA was isolated from WT rice supplied with 0.5/2.5N: 0.5/2.5 mM NO<sup>3</sup> <sup>−</sup> and 0.5/2.5 A:0.5/2.5 mM NH<sup>4</sup> <sup>+</sup> as an N source for 2 weeks. (A) relative expression of OsNRT2.1/OsNRT2.3/OsNAR2.1 and (D) OsNRAMP3/OsNRAMP5/OsNRAMP6 in leaves; (B) relative expression of OsNRT2.1/OsNRT2.3/OsNAR2.1 and (E) OsNRAMP3/OsNRAMP5/OsNRAMP6 in sheath; (C) relative expression of OsNRT2.1/OsNRT2.3/OsNAR2.1 and (F) OsNRAMP3/OsNRAMP5/OsNRAMP6 in roots. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between different N treatments (P < 0.05, one-way ANOVA). FIGURE S3 \| Mn concentration in seeds and husk of OsNRT2.1/OsNRT2.3b overexpression lines. Mn concentrations in seeds of (A) OsNRT2.1 overexpression lines, (B) OsNRT2.3b overexpression lines and (C) OsNRT2.3a overexpression lines. Mn concentration in husk of (D) OsNRT2.1 overexpression lines, (E) OsNRT2.3b overexpression lines and (F) OsNRT2.3a overexpression lines. b-O1/2/8: three OsNRT2.3b overexpression lines; a-O1/2: two OsNRT2.3a overexpression lines. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between N treatments (P < 0.05, one-way ANOVA). FIGURE S4 \| Identification of transgenic lines. (A) Southern blot of genomic DNA isolated from WT and transgenic plants. Hybridization was performed using a hygromycin gene probe. P, positive control; M, marker. Extraction of total RNA from roots and shoots of WT and transgenic lines and qRT-PCR results under. (B) M: DNA molecular-weight marker II, DIG – labeled; P: positive controls. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between N treatments (P < 0.05, one-way ANOVA). FIGURE S5 \| Comparison of total N/Mn concentrations and content of transgenic plants at different nitrogen supply levels. (A–C) Under 0.5 mM NH<sup>4</sup> <sup>+</sup> treatments, (A) total N concentration, (B) total N content and (C) Mn content of roots and shoots. (D–F) Under 0.5 mM NO<sup>3</sup> <sup>−</sup> treatments, (D) total N concentration, (E) total N content and (F) Mn content of roots and shoots. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between WT and overexpression lines (P < 0.05, one-way ANOVA). FIGURE S6 \| Assessment of the length and width of seeds in different lines under WL and AWD treatments. (A) Seeds lengths (mm), (B) seed widths (mm). Error bars: standard error (n = 4 plants), 15 repeats. Different letters indicate a significant difference between WT and overexpression lines (P < 0.05, one-way ANOVA). FIGURE S7 \| Effects of different irrigation conditions on Mn concentrations in rice husk. Under WL and AWD, (A) total N concentration and (B) Mn concentration of rice husk were assessed. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between the irrigation conditions of all lines (P < 0.05, one-way ANOVA). FIGURE S8 \| Effects of different irrigation conditions on other elements in rice seeds. Under WL and AWD, Fe and Mg concentrations of husk (A,B) and seeds (C,D). Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between WT and overexpression lines in different irrigation conditions (P < 0.05, one-way ANOVA). FIGURE S9 \| Diagram of RNA sampling in WT and transgenic plants. FIGURE S10 \| Effect of transgenic lines on total N/Mn content in vegetative organs under AWD conditions. (A) Dry weight of different parts in all lines, (B) Total N concentration, (C) Total N content, (D) NO<sup>3</sup> <sup>−</sup> concentration, (E) Manganese concentration, and (F) Manganese content from different parts of all lines. Error bars: standard error (n = 4 plants). Other leaves: second and third leaves. Different letters indicate a significant difference between WT and overexpression lines (P < 0.05, one-way ANOVA). FIGURE S11 \| Concentration of other elements in different parts of transgenic lines under AWD conditions. (A) Mg concentration, (C) Mg content, (B) Fe concentration, (D) Fe content. Error bars: standard error (n = 4 plants). Different letters indicate a significant difference between WT and overexpression lines (P < 0.05, one-way ANOVA). FIGURE S12 \| Correlation analysis between expression of OsNRT2.1 and total nitrogen/nitrate concentration in flag leaves of wt and transgenic lines. (A) Linear Analysis of relative expression of OsNRT2.1 and total N concentration. (B) Linear Analysis of relative expression of OsNRT2.1 and nitrate concentration. TABLE S1 \| Primers used to amplify the OsNRT2.1 open reading frame. TABLE S2 \| Primers used for quantitative real-time polymerase chain reaction. ### REFERENCES fpls-09-01192 August 13, 2018 Time: 20:0 # 11 metal ATPase OsHMA2. Plant Physiol. 162, 927–939. doi: 10.1104/pp.113.21 6564 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Luo, Chen, Zhu, Liu, Li, Lu, Ye, Xu and Fan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Elemental Profiling of Rice FOX Lines Leads to Characterization of a New Zn Plasma Membrane Transporter, OsZIP7 Felipe K. Ricachenevsky1,2,3 \, Tracy Punshon<sup>3</sup> , Sichul Lee<sup>4</sup> , Ben Hur N. Oliveira<sup>1</sup> , Thomaz S. Trenz<sup>1</sup> , Felipe dos Santos Maraschin<sup>5</sup> , Maria N. Hindt<sup>3</sup> , John Danku<sup>6</sup> , David E. Salt<sup>6</sup> , Janette P. Fett1,5 and Mary Lou Guerinot<sup>3</sup> <sup>1</sup> Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, <sup>2</sup> Departamento de Biologia, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, Brazil, <sup>3</sup> Department of Biological Sciences, Dartmouth College, Hanover, NH, United States, <sup>4</sup> Center for Plant Aging Research, Institute for Basic Science (IBS), Daegu, South Korea, <sup>5</sup> Departamento de Botânica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, <sup>6</sup> School of Biosciences, University of Nottingham, Loughborough, United Kingdom ### Edited by: Edgar Peiter, Martin Luther University of Halle-Wittenberg, Germany #### Reviewed by: Scott Aleksander Sinclair, Ruhr-Universität Bochum, Germany Massimiliano Corso, Free University of Brussels, Belgium Damien Blaudez, Université de Lorraine, France \Correspondence: Felipe K. Ricachenevsky [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 21 February 2018 Accepted: 04 June 2018 Published: 03 July 2018 #### Citation: Ricachenevsky FK, Punshon T, Lee S, Oliveira BHN, Trenz TS, Maraschin FS, Hindt MN, Danku J, Salt DE, Fett JP and Guerinot ML (2018) Elemental Profiling of Rice FOX Lines Leads to Characterization of a New Zn Plasma Membrane Transporter, OsZIP7. Front. Plant Sci. 9:865. doi: 10.3389/fpls.2018.00865 Iron (Fe) and zinc (Zn) are essential micronutrients required for proper development in both humans and plants. Rice (Oryza sativa L.) grains are the staple food for nearly half of the world's population, but a poor source of metals such as Fe and Zn. Populations that rely on milled cereals are especially prone to Fe and Zn deficiencies, the most prevalent nutritional deficiencies in humans. Biofortification is a cost-effective solution for improvement of the nutritional quality of crops. However, a better understanding of the mechanisms underlying grain accumulation of mineral nutrients is required before this approach can achieve its full potential. Characterization of gene function is more time-consuming in crops than in model species such as Arabidopsis thaliana. Aiming to more quickly characterize rice genes related to metal homeostasis, we applied the concept of high throughput elemental profiling (ionomics) to Arabidopsis lines heterologously expressing rice cDNAs driven by the 35S promoter, named FOX (Full Length Over-eXpressor) lines. We screened lines expressing candidate genes that could be used in the development of biofortified grain. Among the most promising candidates, we identified two lines ovexpressing the metal cation transporter OsZIP7. OsZIP7 expression in Arabidopsis resulted in a 25% increase in shoot Zn concentrations compared to non-transformed plants. We further characterized OsZIP7 and showed that it is localized to the plasma membrane and is able to complement Zn transport defective (but not Fe defective) yeast mutants. Interestingly, we showed that OsZIP7 does not transport Cd, which is commonly transported by ZIP proteins. Importantly, OsZIP7-expressing lines have increased Zn concentrations in their seeds. Our results indicate that OsZIP7 is a good candidate for developing Zn biofortified rice. Moreover, we showed the use of heterologous expression of genes from crops in A. thaliana as a fast method for characterization of crop genes related to the ionome and potentially useful in biofortification strategies. Keywords: zinc, ZIP transporter, rice, fox lines, synchrotron x-ray fluorescence, ionomics, biofortification ### INTRODUCTION fpls-09-00865 June 30, 2018 Time: 16:14 # 2 Zinc (Zn) is an essential micronutrient for plant nutrition and development, being a catalytic and structural co-factor in a large number of enzymes and regulatory proteins, including transcription factors (Marschner, 1995; Maret, 2009). However, Zn can become toxic in concentrations above a certain threshold. Fe participates in Fenton chemistry, generating reactive oxygen species. However, Zn competes with other ions for binding sites, and can become toxic in concentrations above a certain threshold (Clemens, 2001; Briat, 2002). Thus, plants have to keep Zn concentration within a narrow range for proper function. Many proteins are dedicated to Zn homeostasis, including organ and tissue partitioning as well as subcellular compartmentalization (Ricachenevsky et al., 2015). Zn deficiency is the one the most widespread mineral nutritional disorders in humans, second only to Fe deficiency. Conservative estimates suggest that 25% of the human population is at risk of becoming Zn deficient (Maret and Sandstead, 2006). A diet composed mainly of milled cereal grains, common among poor populations, increases the risk of mineral deficiencies because staple foods, including rice (Oryza sativa), have low concentrations of Zn (as well as Fe) in edible tissues (Gomez-Galera et al., 2010). Biofortification, the increase of nutrient concentrations in edible portions of crops before harvesting, has been proposed as a cost-effective solution for micronutrient malnutrition (White and Broadley, 2005; Murgia et al., 2013). Rice is a staple food for nearly half of the world's population<sup>1</sup> and a model species for monocots, making it an obvious candidate for biofortification efforts. A recent screening of a large diversity panel of rice genotypes indicated that it is possible to breed for Zn concentrations in seeds (Pinson et al., 2014). However, rice grains have lower concentration of Zn compared to other cereals, and thus genetic engineering tools might be useful to generate biofortified plants (Kennedy and Burlingame, 2003; Pfeiffer and McClafferty, 2008). In order to devise strategies for increasing micronutrient concentrations in grains, it is necessary to understand how plants acquire, distribute and store Fe and Zn within their tissues, which proteins are involved in each step and which would be good candidates for targeted, molecular breeding approaches. Despite the knowledge accumulated in recent years (Sperotto et al., 2012; Ricachenevsky et al., 2015), functional characterization of genes related to Fe and Zn homeostasis in rice is slower in comparison to the model species Arabidopsis thaliana. Due to genome size and complexity, and availability of protocols for genetic transformation and mutant generation, gene characterization in other crops such as wheat, maize and barley is even more time-consuming (Wu et al., 2015). Thus, strategies for fast, medium to high-throughput gene characterization would help to identify promising candidates for biofortification. Heterologous expression of crop genes in A. thaliana has been used in several studies to demonstrate gene function. In order to allow high-throughput analyses of interesting phenotypes, more than 30,000 independent A. thaliana lines over-expressing rice genes were developed, and named FOX lines (Full-length Over-eXpressor Arabidopsis lines; (Kondou et al., 2009; Sakurai et al., 2011). These lines have been successfully used for characterization of genes associated with several processes in plants including responses to fungal and bacterial pathogens (Dubouzet et al., 2011), tolerance to abiotic stresses (Yokotani et al., 2008, 2009), enzyme characterization (Higuchi-Takeuchi et al., 2011; Anders et al., 2012) and in metabolomics profiling (Albinsky et al., 2010). More recently, similar approaches were used to describe stress-related genes in the halophyte Eutrema salsugineum (Ariga et al., 2015). In this work, we characterized a collection of Arabidopsis FOX lines expressing rice genes using ionomics techniques in order to demonstrate their feasibility for the rapid functional characterization of crop genes with potential use in biofortification strategies. By combining elemental profiling by inductively-coupled plasma mass spectrometry (ICP-MS; (Salt et al., 2008) and Synchrotron X-Ray fluorescence (SXRF; Punshon et al., 2013), we described lines that express a new plasma membrane Zn transporter of the Zn-regulated, ironregulated transporter-like protein (ZIP) family from rice, OsZIP7. Our work demonstrates that ionomics of A. thaliana lines heterologously expressing rice cDNAs is a useful method for the rapid characterization of genes involved in regulation of the ionome, an approach that should also be feasible for other crops. ### MATERIALS AND METHODS ### Plant Materials and Growth Conditions For ionomics profile screening, Rice FOX lines and Col-0 WT seeds were sown and cultivated as described (Lahner et al., 2003), with minor modifications. After 3 days at 4 ◦C for stratification, trays were kept in a climate-controlled growth room with 10 h of light (90 µmol.m−<sup>2</sup> s −1 )/14 h dark, humidity of 60% and temperature ranging from 19 to 22◦C. Twelve plants of each genotype, including WT Col-0, were cultivated for 6 weeks, and were watered twice a week with 0.25X Hoagland solution using 10 µM Fe-HBED [N,N<sup>0</sup> -di(2-hydroxybenzyl) ethylenediamine- N,N<sup>0</sup> -diacetic acid monohydrochloride hydrate; Strem Chemicals, Inc.] as the Fe source. For growth in axenic conditions, seeds were sterilized for 15 min in 1.5% sodium hypochloride with 0.05% SDS, washed five times in sterile H2O and stratified at 4◦C for 3 days. Sterile 0.1% agar was used to suspend seeds, which were sown using a pipette onto plates made with full strength Gamborg's B5 media plus vitamins, 1 mM MES [2-(N-morpholino)ethanesulfonic acid], 2% sucrose and 0.6% agar. After 5 days, seedlings were transferred to minimal media containing 2 mM MES, 2 mM Ca(NO3)2.4H2O, 0.75 mM K2SO4, 0.65 mM MgSO4.7H2O, 0.1 mM KH2PO4, 10 µM H3BO3, 0,1 µM MnSO4, 50 nM CuSO4, 5 nM (NH4)6Mo7O<sup>24</sup> and 50 µM Fe-EDTA. ZnSO<sup>4</sup> was added to a final concentration of 50 nM in control conditions, or at indicated concentrations. Seedlings were analyzed after 15 days <sup>1</sup>http://www.fao.org/rice2004/en/rice-us.htm of growth and plates were kept at 22◦C with 16 h of light/8 h of dark in growth chambers. For ICP-MS analyses of seed samples, plants were grown on soil in a growth room at 22◦C with 16 h of light/8 h of dark. Seeds were collected from five plants of each genotype and analyzed by ICP-MS as above. ### Elemental Analyses by ICP-MS Elemental concentration analyses of leaf samples were performed as described (Lahner et al., 2003), with the minor modification that the plants were grown in soil for 6 weeks. Sample handling and preparation was performed as described (Lahner et al., 2003). Data was normalized across different trays Col-0 values, which were present in each tray. All data is publicly available at www. ionomics.org for download. For ICP-MS analyses of shoots and roots of axenically grown plants, metals were desorbed from samples for 10 min on ice with cold 5 mM CaSO4, 1 mM MES, pH 5.7, for 5 min on cold 5 mM CaSO4, 10 mM EDTA, 1 mM MES, pH 5.7 and then washed twice with cold ultrapure water (Haydon et al., 2012). Sample processing was performed as above. We used 12 replicates por line for the initial screening, five replicates per line for shoots and roots, and eight replicates for seeds ICP-MS analyses. ### Subcellular Localization For protoplast preparation, A. thaliana Col-0 plants were grown in soil in a growth chamber at 22◦C with 12 h of light/12 h of dark. After 4 weeks, approximately 25 leaves were detached and had their abaxial epidermis removed following digestion by the tape-sandwich method (Wu et al., 2009). Macerozyme and cellulase treatment and protoplast recovery from the remaining leaf mesophyll were performed as described (Yoo et al., 2007). For subcellular localization in protoplasts, the OsZIP7 coding sequence lacking the stop codon was amplified using specific primers (Supplementary Table S2) and cloned into the pENTR/D-TOPO entry vector. Subsequently, LR recombination was performed into a vector containing a C-terminal fusion with YFP, pEarleyGate101, generating pEarleyGate101-OsZIP7. High concentrations of the final construct were prepared using PureYieldTM Plasmid Midiprep from Promega <sup>R</sup> . AtAHA2-RFP construct (Kim et al., 2001) was used as plasma membrane localization control. Protoplast transfection was performed as described (Yoo et al., 2007). Because of the large size of the pEarleyGate101- OsZIP7 construct, 20 µg of DNA were used. For AtAHA2-RFP, 10 µg were used. For visualization of YFP and RFP signals, a Nikon Eclipse Ti inverted microscope stand was used, and image capture and processing was performed with Nikon Elements software. Nicotiana benthamiana plants were grown in a growth chamber at 24◦ C in a 16/8 h light/dark cycles until leaves were fully expanded for agroinfiltration. Transient expression in N. benthamiana leaves was performed as described previously (Sparkes et al., 2006). Agrobacterium tumefaciens (EHA105 strain) carrying pEarleyGate101-OsZIP7 binary vector was coinfiltrated with Agrobacterium tumefaciens carrying pBIN20/PM-CK binary vector, which contains the coding sequence of PIP2A of A. thaliana fused in frame with cyan fluorescent protein (CFP; Nelson et al., 2007), in an optical density ratio of 1:1. Plasmolysis was induced using a 20% NaCl hypertonic solution. Fluorescence microscopy was performed under an Olympus FV1000 confocal laser-scanning microscope, using YFP and CFP filters. Images were captured with a high-sensitivity photomultiplier tube detector. Due to confocal microscope limitation in both co-localization experiments, we obtained subsequent images showing fluorescence signals from the same cells, without the signal overlay. ### Yeast Assays A full-length version of OsZIP7 was amplified using specific primers (Supplementary Table S1) and cloned into the pDR195 vector using XhoI and BamHI sites. As a control, AtIRT1 was also amplified and cloned into pDR195 using XhoI and BamHI sites. Yeast strains BY4743 (MATa/α his311/his311 leu210/ leu210 LYS2/lys210 met1510/MET15 ura310/ura310), ZHY3 (MATα ade6 can1 his3 leu2 trp1 ura3 zrt1::LEU2 zrt2::HIS3) and DEY1453 (MATa/MATα ade2/ADE2 can1/can1 his3/his3 leu2/leu2 trp1/trp1 ura3/ura3 fet3-2::HIS3/fet3-2::HIS3 fet4- 1::LEU2/fet4-1::LEU2) were grown in YPD media pH 5.3 (DEY1453 was grown in pH 4 to increase Fe availability) and transformed with pDR195, pDR195-OsZIP7, or pDR195-AtIRT1 by the LiOAc/PEG method (Gietz and Schiestl, 2007). Selection of transformants was performed in SD media without uracil (SD –ura: 6.7 g/L yeast nitrogen base without amino acids, supplemented with 2% glucose, 0.1% casamino acids, 0.01% adenine, and 0.01% tryptophan), pH 5.3. Colonies were grown overnight in liquid SD -ura media, diluted to OD<sup>600</sup> 1.0, 0.1, 0.01 and 0.001, and spotted onto plates. To test for Cd toxicity, SD ura was amended with CdCl<sup>2</sup> at given concentrations. To test for Zn deficiency, no Zn was added, and 10 µM ZnCl<sup>2</sup> was added to control plates. To test for Fe deficiency, the pH was raised to 6.0, and compared to control plates at pH 5.3. Pictures were taken after 3–5 days of growth. ### Synchrotron X-Ray Fluorescence For microtomography of seed, tomograms were collected at the bending magnet beamline X26A at the National Synchrotron Light Source, Brookhaven National Laboratory. µ-SXRF seed analyses were performed as described (Kim et al., 2006), using Col-0 and OsZIP7-FOX1 seeds deved from plants grown simultaneously. Elemental abundances (weight fraction) were calculated for the fluorescence measurements as described (McNear et al., 2005). ### Statistical Analyses For ionomics profile comparison between FOX lines and Col-0, we used intra-tray comparisons (i.e., each line had their profile compared to Col-0 plants growing in the same tray). Concentration values for a given element (x) were considered outliers when x > Q75% + 1.5 × Q75% − Q25% or x < Q25% − 1.5 × Q75% − Q25%, where Q75% − Q25% represents 50% of the values observed (i.e., between the 1st and 3rd quartile). Statistical significance was accessed using the Wilcoxon–Mann–Whitney test and the Benjamini–Hochberg correction. All other data were subjected to ANOVA and means were compared by the Tukey HSD test. ### RESULTS ### Rice FOX Lines Selection and Elemental Analysis To perform an informed selection of Rice FOX lines, we searched the rice genome<sup>2</sup> for predicted proteins with similarity to proteins described in the literature as involved in Zn and Fe homeostasis in plants. We used sequences from known genes families as queries, such as ZIP (Zinc-Regulated/Iron-Regulated Transporter Protein; Eide et al., 1996), YSL (Yellow Stripe-Like; Lee et al., 2009), ZIFL (Zinc-Induced Facilitator-Like; Haydon and Cobbett, 2007; Ricachenevsky et al., 2011), MTP (Metal Tolerance Protein; Ricachenevsky et al., 2013b), NRAMP (Natural Resistance Associated Macrophage Protein; Sasaki et al., 2012), OPT (Oligopeptide Transporter; Stacey et al., 2008), VIT (Vacuolar Iron Transporter; Zhang et al., 2012), FER (Ferritins; Stein et al., 2009), PCS (Phytochelatin Synthase; Li et al., 2007), transcription factors of the NAC (Non-Apical Meristem/Arabidopsis Transcription Activation Factor/Cup-Shaped Cotyledon) stress-related subfamily (Ricachenevsky et al., 2013a), IRO2 (Iron-related transcription factor 2; Ogo et al., 2006), and enzymes of the phytosiderophore biosynthetic pathway (Deoxymugineic acid synthase – DMAS; Bashir et al., 2006). Characterized genes for each family cited above were selected and used as queries to search the rice genome. All rice gene products showing at least 30% similarity to query sequences were compiled and used as queries to search the Rice FOX <sup>2</sup>http://rice.plantbiology.msu.edu/ Database<sup>3</sup> (Sakurai et al., 2011). We identified 42 lines expressing 24 different rice genes, comprising 13 different gene families (Supplementary Table S1). Fifteen genes were expressed in two or more of the FOX lines, while nine were expressed in a single line (Figure 1). All FOX lines were grown under the same conditions alongside WT Col-0, in soil amended with subtoxic concentrations of trace elements, watered with Hoagland solution, and after 6 weeks leaves were collected to quantify 20 elements by ICP-MS (Lahner et al., 2003). Comparing the ionomics profiles of each FOX line with WT, we sought to find statistically significant differences in elemental concentrations (Figure 1). We found two lines expressing OsZIP7 that showed a consistent 25% increase in leaf Zn concentration each (Figure 1, lines K11313\_OsZIP7 and K27616\_OsZIP7). It is important to note the initial screen was performed in segregating FOX lines. We would expect changes in elemental profiles of FOX lines to be dominant, as they are a result of heterologous expression using 35S promoter. Since we analyzed 12 individual plants per line, we expected to find 3 wild types on average for each line, which would allow to detect significant changes in the ionome. Indeed, we demonstrated the feasibility of performing such a screen in FOX lines before the additional time required for isolating homozygous lines. We further confirmed the elemental profile phenotype of OsZIP7-FOX lines in the next generation (hemizygous lines; Figure 2) and decided to further characterize the molecular function of OsZIP7. ### OsZIP7 Can Complement Yeast Cells Defective in Zn Uptake We expressed the OsZIP7 full-length coding sequence in different yeast mutant strains to assess its metal transport ability. When introduced into the Zn uptake-defective zrt1zrt2 mutant, OsZIP7 was able to rescue growth in low Zn medium (Figure 3). When <sup>3</sup>http://ricefox.psc.riken.jp/ expressed in the Fe uptake-defective strain fet3fet4, however, OsZIP7 did not restore growth in high pH medium, which lowers Fe availability (Supplementary Figure S1), indicating that OsZIP7 is able to transport Zn but not Fe. This is in contrast to a previous report of OsZIP7 as an Fe transporter (Yang et al., 2009). We also transformed the wild-type strain BY4743 and tested whether OsZIP7 increases cadmium (Cd) toxicity, indicative of Cd transport ability. When growing in media containing 50 µM Cd, both OsZIP7 and empty vector-transformed yeast were able to grow, while AtIRT1-transformed cells grew to a lesser extent (Figure 3C). Therefore, we concluded that the OsZIP7 protein is likely to function as Zn transporter, but not as an Fe or Cd transporter. ### OsZIP7 Is Localized at the Plasma Membrane in A. thaliana Protoplasts and N. benthamiana Epidermal Cells In order to determine the subcellular localization of OsZIP7, we transiently expressed an OsZIP7-YFP construct in A. thaliana protoplasts, either alone or co-transfected with AHA2-RFP, a known plasma membrane marker. The OsZIP7-YFP signal was observed in a pattern that indicated plasma membrane localization (Figure 4). When co-expressed with the AHA2-RFP control, (Kim et al., 2001), expression of OsZIP7-YFP and AHA2- RFP were localized in a similar pattern, although it is possible that OsZIP7 also localized to internal membranes (Figure 4). We also transiently expressed the OsZIP7-YFP construct in Nicotiana benthamiana epidermal cells. N. benthamiana leaves were co-agroinfiltrated with the plasma membrane marker PIP2A-CFP (cyan fluorescent protein, Nelson et al., 2007). OsZIP7-YFP and PIP2A-CFP localization is highly similar in cells co-expressing both constructs (Figure 5). When plasmolyzed, colocalization of OsZIP-YFP with the plasma membrane marker was also evident (Figure 5). Plasma membrane localization is consistent with our yeast complementation results, since OsZIP7 complemented the zrt1zrt2 mutant (Figure 3), which lacks two ZIP plasma membrane transporters (MacDiarmid et al., 2000). Thus, OsZIP7 is likely to be a Zn transporter localized at the plasma membrane. ### Expression of OsZIP7 in Arabidopsis Leads to Enhanced Zn Sensitivity and Disruption of Zn Root-to-Shoot Partitioning To gain more information on OsZIP7 function, we tested the Zn sensitivity of two independent homozygous OsZIP7-FOX lines (OsZIP7-FOX1, derived from FOX lines K11313, and OsZIP7-FOX2, derived from FOX line K27616) grown on media containing excessive Zn levels. When both OsZIP7-FOX lines were grown at control conditions, we observed similar growth compared to wild type lines (WT; Figure 6A). However, at 100 µM Zn, both OsZIP7-FOX lines showed decreased growth, with significantly decreased root length and shoot fresh weight compared to wild type. At 200 µM Zn, OsZIP7-FOX lines were stunted, with short roots and small shoots (Figure 6A). Root length was 30–35% decreased in OsZIP7-FOX lines compared to wild type in 100 and 200 µM Zn, while shoot fresh weight was about 40% decreased in 100 µM Zn and 60– 65% in 200 µM Zn (Figures 6B,C). Thus, we concluded that OsZIP7 expression in Arabidopsis leads to increased sensitivity to Zn. We also quantified elemental concentration by ICP-MS in roots and shoots of wild type and OsZIP7-FOX1 plants under the same conditions, as well as in plants grown at 50 µM (the highest non-toxic Zn concentration, in our growth conditions). Zn concentrations were significantly higher in leaves of OsZIP7-FOX plants grown in media containing 50, 100, and 200 µM Zn compared to wild type (Figure 7A). When comparing Zn concentrations in roots of WT and OsZIP7- FOX1, the opposite effect was observed, with OsZIP7-FOX1 having lower Zn concentrations than wild type, especially under 200 µM Zn, in which OsZIP7-FOX1 root Zn concentrations were only 40% of wild type (Figure 7B). To clarify the change in Zn partitioning caused by expression of OsZIP7-FOX1, we compared the shoot-to-root ratio of WT and OsZIP7 plants. Clearly, ectopic expression of OsZIP7 throughout the plant led to increased root-to-shoot translocation of Zn (Figure 7C). Interestingly, changes in Fe concentrations were also seen in both roots and shoots of OsZIP7-FOX1 plants: roots of OsZIP7- FOX1 plants had higher Fe concentrations than in the WT when grown under 100 and 200 µM Zn, and shoots had higher Fe concentrations when grown on 200 µM Zn (Supplementary Figure S2). ### OsZIP7 Over-Expression Leads to Zn Accumulation in Seeds Because we are interested in good candidates for biofortification of the edible parts of plants, we decided to investigate the effect of OsZIP7 expression on Arabidopsis seed metal accumulation and distribution. We performed ICP-MS elemental quantification of WT and OsZIP7-FOX seeds from both lines. Zn concentration was 20–25% higher in the OsZIP7-FOX plants than in WT (Figure 8A), an increase similar to what was observed in leaves of soil-grown plants by ICP-MS (Figure 2). Interestingly, we also observed a small but significant decrease in Cd concentration in OsZIP7-FOX seeds, especially in OsZIP7-FOX2 line, a trait that is desirable when considering OsZIP7 as a candidate for biofortification (Supplementary Figure S3). The same trend was observed for Cu concentration (Supplementary Figure S3). We also used synchrotron X-ray fluorescence (SXRF) microtomography to directly visualize metal distribution and abundance in seeds. Zn was clearly more abundant in OsZIP7- FOX seeds compared to WT (about twice as much), but there were no changes in distribution (Figure 8B). Abundance of other elements (K, Ca, Mn, Fe, and Cu) did not vary, or varied only slightly (Figure 8C). As we have not observed changes in these elements concentration by ICP-MS, except for OsZIP7- FOX2 line which had a slightly decrease in Cu (Supplementary Figure S3), it is possible that the observed differences are seedto-seed variation. These results indicate that OsZIP7 constitutive expression increases Zn concentration in Arabidopsis seeds and slightly reduces Cd concentration, indicating OsZIP7 is a good candidate for Zn biofortification. ### DISCUSSION ### Coupling Rice FOX Lines and Ionomics Profiling Is a Fast Method for Identification of Metal-Related Genes From Rice Numerous proteins have been described as having a role in metal homeostasis in plants, including transporters, transcription factors and enzymes (for reviews, Hindt and Guerinot, 2012; Sinclair and Kramer, 2012; Sperotto et al., 2012; Ricachenevsky et al., 2015). However, gene characterization in crops is not as fast as in A. thaliana. There is need to translate the information from models to agronomically important plants. In rice, many transporters already annotated in the genome do not have an assigned molecular function, and characterization of possible targets for biofortification in other economically relevant cereals such as corn (Zea mays), sorghum (Sorghum bicolor), and wheat (Triticum aestivum) is difficult. Thus, the use of heterologous systems for high-throughput characterization of genes from species that are slower to cultivate or especially difficult to transform is attractive. The use of Rice FOX lines was successful to describe proteins involved in several processes (Yokotani et al., 2008, 2009; Albinsky et al., 2010; Dubouzet et al., 2011; Higuchi-Takeuchi et al., 2011; Anders et al., 2012). In this work, we highlight the feasibility of using FOX lines coupled with ionomics profiling for characterization of metal-related genes from crop species, such as rice. Besides OsZIP7, which we discussed in detail, our screen identifies other examples of interesting lines that might be studied in depth to understand their role in the regulation of the ionome. The results reported here are derived from a subset of FOX lines selected because they contain cDNAs from gene families involved in metal homeostasis. Similar focused approaches have successfully identified genes from E. salsugineum that confer heat or heat and salt stress tolerance when expressed in A. thaliana, in which 78 and 433 lines were tested, respectively (Higashi et al., 2013; Ariga et al., 2015). In these studies, T2 generations were also screened for stress tolerance. Thus, it is clear that phenotyping of FOX lines and similar tools can be performed even without isolation of homozygous lines, since it is expected that altered phenotypes would be dominant. It should be considered that an unbiased screen (i.e., not focused on selected metal transporters) lines expressing heterologous genes could lead to the identification of previously unknown regulators of the ionome. Moreover, it should also be noted that constitutive expression of a gene in a heterologous system might overcome regulatory mechanisms that might modulate protein activity (i.e., post-transcriptional regulation) in their native environment, increasing the chances of identifying interesting genes that could otherwise be regulated by transcriptional and post-transcriptional mechanisms. ### OsZIP7 Is a New Zn Transporter The first member of the ZIP (Zinc-regulated/Iron-Regulated Protein) family of transporters described was AtIRT1 (Eide et al., 1996), followed by characterization of several ZIP members in Arabidopsis, rice, corn, barley (Hordeum vulgare) among other species (Lee et al., 2010a,b; Li et al., 2013; Milner et al., 2013; Tiong et al., 2014). Plants harbor many ZIP genes in their genomes, with as many as 16 loci in the genomes of some Poaceae (Tiong et al., 2015). ZIP transporters are known for having broad substrate specificity: AtIRT1 is able to transport Zn+<sup>2</sup> , Fe2+, Mn2+, Cd2+, Co2+, Ni2+, and Fe3<sup>+</sup> (Korshunova et al., 1999) while its rice ortholog OsIRT1 transports Fe2+, Zn2+, and Cd2<sup>+</sup> (Ishimaru et al., 2006; Lee and An, 2009). AtIRT2 and AtIRT3 transport Fe2<sup>+</sup> and Zn+<sup>2</sup> , but not Mn2<sup>+</sup> or Cd2<sup>+</sup> (Vert et al., 2001; Lin et al., 2009). In Arabidopsis, others ZIPs are commonly Zn2<sup>+</sup> or Zn2<sup>+</sup> and Mn2<sup>+</sup> transporters, with AtZIP7 also being able to transport Fe2<sup>+</sup> (Milner et al., 2013). Moreover, ZIP proteins characterized in plants are mostly localized to the plasma membrane, which also seems to be true for OsZIP7 based on our data (Figures 4, 5). OsZIP7 is the closest rice homolog of barley HvZIP7 and maize ZmZIP7 (Tiong et al., 2014; Li et al., 2016) and Arabidopsis AtIRT3 and AtZIP4 (Li et al., 2013; Tiong et al., 2015). Of these, HvZIP7 and AtIRT3 had their subcellular localization determined to be at the plasma membrane (Lin et al., 2009; Tiong et al., 2014). Here we have shown that OsZIP7 was able to complement the Zn-deficient zrt1zrt2 yeast mutant, but not the Fe-deficient fet3fet4 (Figure 3). OsZIP7 has been indicated as the rice ortholog of barley HvZIP7, which was recently characterized as a Zn transporter (Tiong et al., 2014, 2015). HvZIP7 was localized to the plasma membrane and increased plant Zn root-to-shoot translocation when compared to WT controls in over-expressing barley plants (Tiong et al., 2014). This is consistent with our observation that OsZIP7 expression in A. thaliana under the control of 35S promoter led to increased Zn concentrations in leaves and seeds and increased root-to-shoot Zn translocation (Figures 7, 8). Expression of OsZIP7 in Arabidopsis led to increased rootto-shoot Zn translocation when plants are exposed to high Zn in the growth media, with roots of OsZIP7-FOX lines showing lower Zn concentrations compared to WT, whereas shoots have increased Zn concentrations (Figure 7). This is similar to what was observed for HvZIP7 over-expression in barley, with plants showing higher Zn concentration in shoots and lower in roots compared to null-segregant lines (Tiong et al., 2014). In both our OsZIP7-FOX lines and in HvZIP7 over-expressing plants, Zn concentrations in shoots and leaves were not changed under control conditions. One possible explanation for these phenotypes is that OsZIP7 expression might increase sink strength in shoots, while also causing increased primary Zn uptake in shoots. Zn xylem-loading transporters such as AtHMA2/AtHMA4 (Hussain et al., 2004) may not limit Zn translocation to shoots under such conditions. Although the precise mechanism is not clear, OsZIP7 expression in Arabidopsis and HvZIP7 over-expression in barley seem to result in distinct phenotypes compared to over-expression of other ZIP transporters such as OsZIP4, OsZIP5, and OsZIP8 (Ishimaru et al., 2007; Lee et al., 2010a,b). In seeds, however, OsZIP7- FOX lines and HvZIP7 over-expressing lines showed increased Zn concentrations even without excessive Zn in the media, indicating that higher Zn accumulation for biofortification using OsZIP7/HvZIP7 may not require Zn addition (Figure 8, Tiong et al., 2014). Interestingly, two OsZIP7 protein sequences have been reported in rice, differing in only four amino acid positions: OsZIP7, which is characterized in this work, and OsZIP7a, characterized by Yang et al. (2009). Three aminoacid changes are in positions outside transmembrane domains, while one is inside the VII domain. OsZIP7a was shown to not complement Zn-defective yeast mutants (Yang et al., 2009). HvZIP7 failed to complement the yeast strain zrt1zrt2, although several other lines of evidence indicate its function as a Zn transporter (Tiong et al., 2014). Here we showed that OsZIP7 is able to rescue the zrt1zrt2 yeast mutant phenotype to some extent (Figure 3). Considering the increased Zn sensitivity of Arabidopsis expressing OsZIP7 (Figure 6), these results suggest that OsZIP7 is a Zn transporter. It is possible that OsZIP7 is a low-affinity Zn transporter, as suggested for its closest homologous gene from barley (HvZIP7; Tiong et al., 2014). Interestingly, OsZIP7a has been described as able to complement fet3fet4, suggesting it could transport Fe (Yang et al., 2009). We did not observe fet3fet4 complementation when fet3fet4-expressing OsZIP7 was cultivated in high pH media, while AtIRT1-expressing yeast was able to grow (Figure 3), indicating that OsZIP7 does not transport Fe. However, it is still possible that OsZIP7 transports Fe. One hypothesis is that transport is dependent on pH, with high pH decreasing transport function. Despite that, our results support that OsZIP7 is a Zn transporter. The FOX lines expressing OsZIP7 showed increased Fe concentrations in roots and shoots upon high Zn concentration in the growth media (Supplementary Figure S2). This may indicate that OsZIP7 might transport Fe, although it is not clear why Fe concentrations would increase only under high Zn. Heterologous expression in Arabidopsis of OsZIP7 maize ortholog, ZmZIP7, led to increased Fe and Zn concentrations in all tissues, and concomitant upregulation of the Fe uptake regulon, including AtIRT1 (Li et al., 2016). Conversely, HvZIP7 over-expression in barley does not change Fe concentrations in either shoots or roots, even when plants are cultivated under high Zn in the growth media (Tiong et al., 2014). A possible explanation is that high Zn concentrations induced Fe-deficiency and Fe uptake genes, leading to increased root and shoot Fe concentrations (Supplementary Figure S2). Thus, it is more likely that OsZIP7 is not able transport Fe. Still, future work should address if OsZIP7 and OsZIP7a differ in their substrates and if the four distinct aminoacids can change metal specificity (Yang et al., 2009). ### Potential of OsZIP7 for Zn Biofortification of Rice Seeds OsZIP7 expression in Arabidopsis increased Zn concentration in seeds by 25% (Figure 8). Similarly, HvZIP7 over-expression in barley led to significant increase in Zn concentration in grains, with no changes in other elements (Tiong et al., 2014), whereas expression of ZmZIP7 in Arabidopsis led to increased Zn and Fe concentrations in seeds. Interestingly, we have also observed a decrease in Cd concentration of 12–24% in seeds (Supplementary Figure S3), indicating that OsZIP7 overexpression in rice could lead to increase Zn in grains without concomitantly increasing Cd levels. From a biofortification perspective, that makes OsZIP7 a good candidate for genetic engineering, since Cd co-transport when manipulating Zn and Fe transport such as the ZIP family members should be considered (Slamet-Loedin et al., 2015). OsZIP7 is highly expressed in developing grains in rice plants (Supplementary Figure S4). Several different genes have been used to improve Zn concentration in rice grains, and increases have been moderate so far (for a review, see Ricachenevsky et al., 2015). Two successful transgenic approaches involved activation tagging or over-expression of nicotianamine synthase (NAS) genes (Johnson et al., 2011; Lee et al., 2011). Presumably, increased levels of nicotianamine in these plants facilitate Zn loading in the phloem and translocation to grains, but increased available Zn for translocation might lead to further accumulation. Thus, there is still potential to increase Zn levels. Either OsZIP7 overexpression as a single transgene, combined with OsNAS2 of expressed in specific cell types such as the endosperm could be promising to generate biofortified rice in the future. ### CONCLUSION We have demonstrated that Arabidopsis lines generated to heterologously express rice genes are useful for fast screening genes that are involved in metal homeostasis when combined with elemental analyses. We have also molecularly characterized OsZIP7, a Zn plasma membrane-localized transporter from rice. Based on our results, OsZIP7 is a good candidate for overexpression in rice to generate lines that are able to accumulate Zn in their seeds. ### AUTHOR CONTRIBUTIONS FKR, TP, DES, JPF, and MLG designed the experiments. FKR, TP, and MNH performed the experiments. FKR, TP, BHNO, and JD performed the analyses. FKR, TP, SL, BHNO, TST, FSM, MNH, DES, JPF, and MLG wrote the manuscript. All authors approved the manuscript. ### FUNDING The authors would like to thank FAPERGS (Fundação de Amparo à pesquisa do Estado do RS) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for funding. This work was supported by a grant from the National Science Foundation's Plant Genome program to DES, MLG, FKR, and JPF (DBI 0701119). Use of NSLS facility was supported by the Department of Energy under Contract DE-AC02-98CH10886. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) granted a fellowship to FKR and a research grant to JPF. ### ACKNOWLEDGMENTS The authors would like to thank Brett Lahner and Elena Yakubova for technical assistance for plant growth and ICP-MS analyses, and Prof. Dr. Marcia Maria Auxiliadora Naschenveng Pinheiro-Margis for giving access to laboratory facilities. The authors would also like to especially thank John Danku, which sadly passed away during the preparation of the manuscript. John premature death is a great loss to science and the field of ionomics. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00865/ full#supplementary-material FIGURE S1 \| Yeast phenotype complementation assays. Empty pDR195 vector, pDR195-OsZIP7 or pDR195-AtIRT1 constructs were transformed into yeast cells. Liquid cultures were diluted as indicated before plating. (A) Fe-uptake defective strain fet3fet4 transformed with each construct growing under Fe-sufficient (pH 5.3) or Fe-deficient (pH 6.0) conditions. (B) Wild type strain BY4743 transformed with each construct growing under control or 50 µM Cd conditions. FIGURE S2 \| Metal concentrations in shoots (A–D) and roots (E–H) of Col-0 (white bars) and OsZIP7-FOX Arabidopsis plants (gray bars) grown for 15 days in Minimal Media containing 50, 100, or 200 µM Zn (n = 5). Concentrations of Mn (A,E), Fe (B,F), Cu (C,G), and Cd (D,H) are shown. Different letters show significant differences by ANOVA and Tukey HSD. FIGURE S3 \| Metal concentrations in seeds of Col-0 and OsZIP7-FOX1 and OsZIP7-FOX2 plants (n = 8). Concentrations of Mn (A), Fe (B), Cu (C), and Cd (D) are shown. Different letters show significant differences by ANOVA and Tukey HSD. FIGURE S4 \| Rice OsZIP7 transporter expression pattern based on data from the eFP Browser public database (http://bar.utoronto.ca/efp\_rice/cgi-bin/ efpWeb.cgi). TABLE S1 \| Rice FOX lines used in this work. TABLE S2 \| Primers used in this work. ### REFERENCES fpls-09-00865 June 30, 2018 Time: 16:14 # 11 reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One 6:e24476. doi: 10.1371/journal.pone.0024476 computed microtomography to image metal compartmentalization in Alyssum murale. Environ. Sci. Technol. 39, 2210–2218. doi: 10.1021/es0492034 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Ricachenevsky, Punshon, Lee, Oliveira, Trenz, Maraschin, Hindt, Danku, Salt, Fett and Guerinot. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Genetic Basis and Breeding Perspectives of Grain Iron and Zinc Enrichment in Cereals Ana Luisa Garcia-Oliveira<sup>1</sup> \, Subhash Chander <sup>2</sup> , Rodomiro Ortiz <sup>3</sup> \, Abebe Menkir <sup>1</sup> and Melaku Gedil <sup>1</sup> 1 International Institute of Tropical Agriculture, Ibadan, Nigeria, <sup>2</sup> Department of Genetics & Plant Breeding, Chaudhary Charan Singh Haryana Agricultural University, Hisar, India, <sup>3</sup> Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden Micronutrient deficiency, also known as "hidden hunger," is an increasingly serious global challenge to humankind. Among the mineral elements, Fe (Iron) and Zn (Zinc) have earned recognition as micronutrients of outstanding and diverse biological relevance, as well as of clinical importance to global public health. The inherently low Fe and Zn content and poor bioavailability in cereal grains seems to be at the root of these mineral nutrient deficiencies, especially in the developing world where cereal-based diets are the most important sources of calories. The emerging physiological and molecular understanding of the uptake of Fe and Zn and their translocation in cereal grains regrettably also indicates accumulation of other toxic metals, with chemically similar properties, together with these mineral elements. This review article emphasizes breeding to develop bioavailable Fe- and Zn-efficient cereal cultivars to overcome malnutrition while minimizing the risks of toxic metals. We attempt to critically examine the genetic diversity regarding these nutritionally important traits as well as the progress in terms of quantitative genetics. We sought to integrate findings from the rhizosphere with Fe and Zn accumulation in grain, and to discuss the promoters as well as the anti-nutritional factors affecting Fe and Zn bioavailability in humans while restricting the content of toxic metals. Keywords: biofortification, cereals, iron, zinc, micronutrient deficiency, toxic risks ## INTRODUCTION From the 1950s onwards, the advancement in science and technology together with concerted efforts of international and national agricultural organizations has resulted in significant gains in world food production widely referred to as the "Green Revolution" (Ortiz, 2011). Globally, the availability of sufficient quantities of food is not only a simple achievement of the Green Revolution but has also helped to avert large-scale famines and social and economic upheavals (Khush, 1999). Without the Green Revolution, crop yields in Asia and Latin America would be at least 20% less, food prices would be up 19%, calorie consumption would be down by about 5%, and the number ### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil ### Reviewed by: Ümit Bari ¸s Kutman, Gebze Technical University, Turkey Hamid Khazaei, University of Saskatchewan, Canada #### \Correspondence: Ana Luisa Garcia-Oliveira [email protected] Rodomiro Ortiz [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 16 January 2018 Accepted: 11 June 2018 Published: 02 July 2018 #### Citation: Garcia-Oliveira AL, Chander S, Ortiz R, Menkir A and Gedil M (2018) Genetic Basis and Breeding Perspectives of Grain Iron and Zinc Enrichment in Cereals. Front. Plant Sci. 9:937. doi: 10.3389/fpls.2018.00937 89* Abbreviations: Fe, Iron; Zn, Zinc; CGIAR, Consultative Group on International Agricultural Research; QTL, Quantitative Trait Locus; MAS, Marker-aided Selection; MAB, Marker-aided Breeding; ICP-OES, Inductively Coupled Plasma-Optical Emission Spectrometry. of malnourished children would be up by at least 2% (Evenson and Gollin, 2003). The widespread adoption of these technologies has made it possible to improve the per capita calorie consumption in different continents, especially in the developing world. Overall, the Green Revolution has paid rich dividends in food grain production, particularly cereals, that have led to a significant reduction in the proportion of undernourished people worldwide; however, the problem of malnutrition or lack of quality food still persists, leading to an economic burden for society (Pingali, 2012). Anemia is the most common human nutritional malaise, resulting from iron (Fe) deficiency and affecting 32.9% people worldwide; meanwhile, zinc (Zn) deficiency affects 17% of the world's population, with the highest risk occurring in sub-Saharan Africa and South Asia (Wessells et al., 2012; Kassebaum et al., 2014). In the twenty-first century, there are strong concerns worldwide regarding the ability to produce nutritionally rich food because cereals are inheritably poor in essential micronutrients. Moreover, owing to a burgeoning human population and industrialization, this situation may be further compounded by the production of cereals in areas with low mineral phytoavailability (White and Broadley, 2009). Thus, there is an urgent global need to cope with the problem of micronutrient deficiencies that contribute to what is referred to as "hidden hunger" and affect at least 2 billion people (or 1 out of 3), mostly in sub-Saharan Africa, South Asia, and Latin America (FAO et al, 2015). Diversity in diets in order to provide adequate micronutrient consumption is difficult to achieve in the developing world where resource-poor people cannot afford a variety of different foods. For example, many of the relatively cheap and staple crops such as cereals (Table 1), roots [cassava (Manihot esculentaz)], tubers [sweet potato (Ipomoea batatas) and yam (Dioscorea spp.)], and plantain (Musa spp.) that play a very important role in the daily diets of resource-poor people lack high enough amounts of micronutrients (Gibson et al., 2010). Hence, malnutrition and poor health affect these people, who may suffer from blindness or stunting, and sometimes even face death. To overcome this "hidden hunger," medical supplements and fortification have been pursued (Underwood, 2000). In fact, food fortification has a long history of use in industrialized countries and relies on the addition of micronutrients to processed foods. However, food fortification tends to have a rapid but less sustainable impact, because various safety, technological, and cost considerations may place constraints on such interventions (Allen et al., 2006). Furthermore, such interventions do not always reach the desired target populations (Pfeiffer and McClafferty, 2007). By increasing the micronutrient content of energy-rich crops, micronutrient intakes among the poor can be increased, thereby leading to decreases in the prevalence of micronutrient deficiencies. Biofortification is a strategy that involves the use of plant breeding or agronomic practices to increase the density of essential nutrients in the edible part of staple crops that may help to combat deficiencies among poor people who survive on main staples such as cereals (www.harvestplus.org). Agronomic biofortification is a fertilizer-based approach that relies on soil and/or foliar application of micronutrients either alone or in combination with other fertilizers. It is well-established that a Zn fertilizer strategy is an effective way to biofortify cereal crops with Zn, but recurrent cost is involved (Cakmak and Kutman, 2018). By contrast, genetic biofortification is a seed-based approach that complements agronomic biofortification and also current intervention methods such as supplementation and fortification of foods consumed daily. The aim of this strategy is to enhance the content and bioavailability of micronutrients such as minerals and vitamins in crops through plant breeding, thereby impacting favorably the diets of targeted populations, particularly the resource poor worldwide (Bouis and Welch, 2010). Because of its cost effectiveness, the 2008 Copenhagen Consensus ranked biofortification fifth for combating the world's greatest challenges (http://www.copenhagenconsensus. com/publication/second-copenhagen-consensus-biofortification -best-practice-meenakshi). Biofortifying staple crops through plant breeding is therefore a key option to improve micronutrient deficiency in human diets (Bouis and Saltzman, 2017). ### RELEVANCE OF FE AND ZN TO HUMAN HEALTH Among the mineral nutrients required by humans for their well-being, Fe and Zn play vital roles in numerous metabolic processes and are required in trace amounts by plants as well as animals (Welch and Graham, 2004). For instance, Fe is a well-known essential component of hemoglobin and myoglobin, which are involved in oxygen transport and storage. The greatest effects of Fe deficiency anemia are seen in females during adolescence and pregnancy. Likewise, Fe deficiency affects children's cognitive development until adolescence, and it increases their susceptibility to infectious diseases and mortality (Oliver and Gregory, 2015). Similarly, Zn is also an essential cofactor for many enzymes and regulatory proteins, and it plays an important role in DNA as well as RNA synthesis and gene expression. Children show stunted growth and neurobehavioral difficulties resulting from Zn deficiency, which may increase the incidence and severity of diarrhea, among other conditions (Nriagu, 2007). Furthermore, Zn deficiency seems to be significantly related to anemia associated with Fe deficiency because Zn controls Fe absorption in the intestines (Chang et al., 2010; Graham et al., 2012). Hence, Fe and Zn are acknowledged as outstanding micronutrients owing to their importance in global public health. ### PHYSIOLOGY, GENETICS, AND MOLECULAR ASPECTS OF FE AND ZN ENRICHMENT IN CEREAL GRAINS For genetic biofortification, a better understanding of the key steps of mineral nutrient transport from the rhizosphere to grains is needed, which involves coordination of complex physiological steps such as acquisition of Fe and Zn in roots (uptake), subsequent long-distance transport from roots to shoots, and further redistribution toward the developing seeds (Zhao and McGrath, 2009; Carvalho and Vasconcelos, 2013). Although Fe and Zn are known to accumulate in grains, further insights regarding the underpinning physiology and genetics are yet to be revealed. Soil redox potential and pH affect uptake of Fe by roots and Zn accumulation in grains. Fe is mostly available in the rhizosphere as low solubility Fe3<sup>+</sup> oxyhydrates, while Fe is oxidized in aerobic soils with high pH, thus occurring as insoluble ferric oxides. Free ferric Fe from the oxides become available at low pH for further uptake by roots (Lindsay and Schwab, 1982). Plants have developed different strategies for Fe uptake from the rhizosphere: Strategy I involving ferrous Fe2<sup>+</sup> (non-Poaceae) and Strategy II utilizing ferric Fe3<sup>+</sup> (Poaceae), referred to as reducing and chelating strategies, respectively, or a combination of strategies I and II (Connorton et al., 2017). Poaceae family members such as rice (Oryza sativa L.), maize (Zea mays L.), and wheat (Triticum aestivum L.) follow Strategy II, and their root epidermis secretes phytosiderophores (PSs) that form stable Fe(III) chelates in the rhizosphere (Roberts et al., 2004). TOM1 (Transporter of Mugineic acid family phytosiderophores)/ZIFL4 belongs to the major facilitator superfamily (MFS) that exports Fe3+-PS chelates in rice and barley (Nozoye et al., 2011). From the rhizosphere, these Fe(III)-PS complexes can be taken up into root cells by Yellow Stripe-Like proteins (YSLs). The maize oligopeptide transporter YS1 is the founding member of the YSL family, which facilitates Fe3+-PS complex uptake from the rhizosphere, and subsequently the role of YSL15 has also been confirmed in rice (Curie et al., 2001; Inoue et al., 2009). Besides Strategy II, both rice and barley have a functional homolog of IRT1 (Iron-Regulated Transporter 1) that allows direct uptake of Fe2<sup>+</sup> from the rhizosphere, thus clearly showing the different uptake strategies for Fe2<sup>+</sup> and Fe3<sup>+</sup> in these cereal crops. Soil pH significantly influences Zn acquisition and uptake from the rhizosphere by roots because Zn binds tightly to soil elements and plant cell wall parts under high pH. However, under anaerobic conditions in soils, additional factors such as soil redox potential, total sulfur content, and soluble bicarbonate also affect the availability of Zn (Impa and Johnson-Beebout, 2012). As is also the case for Fe(III), which exhibits even lower solubility, Zn solubilization in the rhizosphere is thought to occur via plantmediated acidification and secretion of low molecular weight organic chelator (Sinclair and Krämer, 2012). Details regarding the role and contribution of Zn acquisition by the plant remain unknown. The uptake of Zn may occur as a divalent cation (Zn2+) or as a Zn-PS complex formed with PSs known as Fe3+ chelators, which are secreted by roots of the plant (von Wiren et al., 1996). ZIP-like transporters may take up Zn as noted in Strategy I plants (Ramesh et al., 2003). Thus, modification of rhizosphere chemistry through root architecture and by secretion of more root exudates that can alter soil pH could be the first promising target for improving Fe and Zn acquisition in cereal roots. After roots acquire Fe and Zn, their translocation to the shoot and further movement to other vegetative organs depend of several steps, going through symplast, xylem, and phloem. Physiological studies have indicated that chelating molecules such as citrate, nicotianamine, and mugineic acid play a vital role in symplast heavy metal homoeostasis including Fe and Zn. Both minerals move through the xylem into the shoot, where Zn can move as free ions or in a complex with organic acids, while Fe is chelated to organic compounds of low molecular weight that are subsequently translocated by the xylem and phloem to other plant organs (Rellan-Alvarez et al., 2010; Lu et al., 2013). In plants, leaves are the most important sink tissue for these minerals where they are required in the plastids and mitochondria for numerous enzymes essential for photosynthesis and other cellular metabolic processes (Gupta et al., 2015). The FDR3 gene has an important role in transporting Fe (Green and Rogers, 2004); while for Zn transport, heavy metal ATPase (HMA), a member of the P1B ATPase family, is a likely candidate for performing this task (Eren and Arguello, 2004: Hussain et al., 2004). At the molecular level, a plethora of genes associated with influx and efflux transporters has been discovered and extensively characterized in plants, including cereals, and which are involved in translocation of these minerals (Kobayashi and Nishizawa, 2012; Ricachenevsky et al., 2015; Vasconcelos et al., 2017). Moreover, transcription factors have also been identified that regulate the genes involved in the uptake of Fe and Zn and synthesis of PSs in cereals. Despite the tremendous improvement in understanding those components participating in translocation, it remains difficult to define precisely the contribution of each of the components in the metal movement flux for each translocation step. Plants remobilize and move nutrients from vegetative source organs into seeds during the filling of grains (Waters and Sankaran, 2011). Hence, the amount of both Fe and Zn in the cereal grain is dependent on the former physiological processes: firstly, their acquisition from the soil by roots, and secondly, transportation to the shoots and further remobilization of stored minerals from leaves when they senesce at grain filling. Despite the large amount of these minerals in the vegetative tissues of cereals, their remobilization from leaves is an important process (from senescence to grain filling), contributing to accumulation in the seeds. Fe and Zn accumulate throughout cereal seeds, being primarily concentrated in the aleurone and embryo parts and to lesser extent in the endosperm, except in rice and barley where Zn appears to be less strictly confined to the aleurone than Fe (Persson et al., 2009). From a biofortification perspective, the heterogeneous distribution of these essential mineral elements in cereal grains further complicates the situation for their efficient loading into the core endosperm (Cakmak et al., 2010). Fe, Zn, copper (Cu), and manganese (Mn) are micronutrients that primarily accumulate in the seed aleurone layer, where phytic acid (the main form of Pi storage in seeds) is a strong chelator of metal cations, binding them to form phytate, a salt of inositol phosphate (Raboy, 2009). However, recent studies have shown that Fe is mainly associated with phytic acid, while Zn is bound to proteins, which clearly suggests that Fe and Zn have a different speciation in cereal grain tissues (Persson et al., 2009; Kutman et al., 2010). Future research, hence, is needed to elucidate the molecular aspects of bivalent metal speciation including Fe and Zn in different tissues of seeds for efficient Fe and Zn biofortification strategies in cereals (Persson et al., 2016). ### BREEDING PERSPECTIVE OF FE AND ZN BIOFORTIFICATION IN CEREALS To address the widespread prevalence of micronutrient deficiency, especially pro-vitamin A, Fe, and Zn, the Consultative Group on International Agricultural Research (CGIAR; https:// www.cgiar.org) HarvestPlus in collaboration with international and national research institutes emphasized biofortification of staple food crops as a cost-effective, easily applicable, and sustainable approach to benefit low-income households. This may complement other efforts aimed at reaching rural populations in developing countries (www.harvestplus.org). Considering the average content of Fe and Zn in cereal grains and their retention after processing, as well as addressing the issues related to the bioavailability of these mineral nutrients, HarvestPlus established target levels of these nutritionally important traits in cereal grains (Table 1). The initial screening of a large amount of crop germplasm suggested the existence of substantial genetic variation for these traits in cereal crops and their wild relatives. Besides the complex nature of these traits, the assays required to measure micronutrient content in plant samples are tedious and costly. While plant breeding has been pursued significantly to achieve biofortification in staple crops, the success in breeding for Fe and Zn biofortification in cereal crops lags behind the development of pro-vitamin A enriched cultivars of staple crops (Andersson et al., 2017). The possible reason for this is a better understanding of the carotenoid biosynthesis pathway in plants, especially in maize, that has led to the deployment of functional markers for pro-vitamin A biofortification in maize (Gebremeskel et al., 2018). Although, there have been significant advances in elucidating the mechanisms related to Fe and Zn homeostasis in model plants, detailed understanding is still lacking. It is noteworthy to highlight that several genes controlling Fe and Zn homeostasis in cereal grains—particularly rice—have been characterized, but their role in genotypic variation for the accumulation of these minerals in the grain remains unclear. Hence, a more holistic breeding approach is required for Fe and Zn biofortification of cereal grains that emphasizes the genetic TABLE 1 \| Information and assumptions used to set target levels for mineral nutrient content in grains of biofortified staple cereals by CGIAR HarvestPlus. Considering 90% retention of both Fe and Zn after processing, and 5 and 25% bioavailability for Fe and Zn, respectively, except Fe in rice grain where bioavailability is 10% (adapted from Bouis and Welch, 2010). enhancement of the contents of these minerals in cereal grains together with the factors that determine their bioavailability in humans such as inhibitors and/or enhancers (Figure 1). Further, there is the need to be cautious regarding inadvertent enhancement of non-essential/certain toxic elements, such as cadmium (Cd), in cereal grains. ### EXPLORING GENETIC VARIABILITY FOR FE AND ZN ENHANCEMENT IN CEREAL GRAINS A pre-requisite for breeding for a specific trait is the availability of its genetic variation within the target gene pool. The task is somewhat complex while breeding for Fe and Zn biofortification in cereal grains because their concentration in the grain depends on various physiological processes. Plant breeders rely on additive genetic effects, transgressive segregation, and heterosis for improving desired traits when enough genetic variation exists. Recently, the genetic variability for these minerals in cereals, particularly maize, rice, wheat, barley (Hordeum vulgare L.), sorghum (Sorghum bicolor L.), and pearl millet (Pennisetum glaucum L.), which are the six most important crops and represent 89% of all cereal production worldwide, was reviewed, and the existence of significant genetic differences for these minerals was reported (Teklic et al., 2013; Goudia and Hash, 2015; Gregory et al., 2017). A survey of 1,400 improved maize genotypes and 400 landraces maintained at the genebank of the International Maize and Wheat Improvement Center (CIMMYT, El Batan, México) indicated about four- to six-fold variation for grain Fe and Zn (Bänziger and Long, 2000). Among the tropical-adapted maize inbreds of the International Institute of Tropical Agriculture (IITA, Ibadan, Nigeria), the best inbreds exhibited 32 to 78% more grain Fe and 14 to 180% more grain Zn over their trial mean (Menkir, 2008). Similarly, several-fold variation for grain Fe and Zn in disomic hexaploid bread wheat has also been reported (Velu et al., 2014; Goudia and Hash, 2015). Correspondingly, substantial variation for these minerals in rice grain has also been reported among different cultivars; however, grain polishing removed up to 50% of the Fe from the brown rice grain (Gregorio et al., 2000; Prom-u-thai et al., 2007). About, two-fold higher Zn concentration but slightly lower Fe concentration was reported in indica rice compared with japonica rice (Yang et al., 1998). Significantly lower Fe and Zn contents were found in the seed of modern cultivars of rice than in landraces (Anandan et al., 2011), thus arguing that breeders failed in introducing quality improvement, particularly for micronutrients, because they gave priority to other traits such as size, shape, and appearance of grain, milling quality, and cooking features. However, a notable aspect of the lower content of these nutrients in the seed of modern cultivars compared with landraces/germplasm may be the yield dilution effect; the total grain nutrient content may not differ significantly between landraces and modern cultivars, and part of this effect could be ascribed to higher grain yield in modern cultivars (Pfeiffer and McClafferty, 2007; McDonald et al., 2008). Therefore, grain yield must be kept in mind when discussing breeding solutions in cereals biofortification. Simple and reliable phenotyping is always preferred by breeders, but an extensive survey of the literature pertaining to the existence of genetic variability for grain Fe and Zn contents in cereal crops clearly suggests that accurate measurement of these mineral nutrients is a challenging task. For the measurement of Fe and Zn content in plants and related material, a wide range of analytical methods is available ranging from semi-quantitative [Perl's Prussian blue and diphenyl thiocarbazone-based dithizone] to fully quantitative [atomic absorption spectrometry, inductively coupled plasma-optical emission spectrometry (ICP-OES), ICP-mass spectrometry, near-infrared reflectance spectrophotometry, X-ray fluorescence spectrometry, elemental distribution maps secondary ion mass spectrometry, synchrotron X-ray, fluorescence spectroscopy, micro-X-ray fluorescence spectroscopy, and Laser-induced breakdown spectroscopy], which can differ substantially with respect to the many attributes describing method performance (Ihnat, 2003; Pfeiffer and McClafferty, 2007). Therefore, the choice of analytical method would depend on the purpose and precision required in estimation. Alternatively, non-destructive quantitative techniques could be the choice method from a breeding perspective, because initial screening for grain Fe and Zn content together with other mineral elements in an appropriately large number of breeding lines can be obtained with minimal or no sample preparation, thereby enabling the discarding of progenies with the lowest content of these mineral elements. There are numerous possibilities for introducing variation in the results of different studies. Firstly, sensitivity of the method used for the quantification of Fe and Zn contents. Secondly, improper postharvest handling of the samples has also been observed to give erroneous results while estimating grain micronutrient concentrations. Furthermore, it is noteworthy that the variability among microenvironments for Fe and Zn may be significant, and most of the research presenting extremely high or low values of these mineral nutrients is based on single-year data; thus, the results are affected by a significant confounded influence of the sampling and environment. Additionally, extremely high trial mean values of these nutrients reported in some studies appear to be affected by the prior use of manure at some locations, because the level of these nutrients in cereal grains, particularly Zn content, can be even lower when grown in infertile/Zndeficient soils (Cakmak et al., 2010; Xu et al., 2011; Velu et al., 2014). ### THE VALUE OF WILD RELATIVES FOR FE AND ZN BIOFORTIFICATION IN CEREALS: OPPORTUNITIES FOR GENETIC GAIN Modern cereal cultivars have a lower concentration of Fe and Zn in grains than landraces. This is because breeding has been mainly aimed at increasing grain yield or improving host plant resistance, among other target traits, instead of also improving the micronutrient concentration in grain. Utilization of landraces or crop wild relatives for genetic gain is not a new concept. The genetic variability for content of micronutrients is becoming acknowledged as a desired trait of crop wild relatives, particularly for rice and wheat. The wild Triticum and Aegilops species have very high grain Fe and Zn contents when compared with both bread and durum wheat (Cakmak et al., 2000; Ortiz-Monasterio and Graham, 2000; Chhuneja et al., 2006; Rawat et al., 2009) as well as synthetic amphiploids (Calderini and Ortiz-Monasterio, 2003). Wild species such as Triticum boeoticum, Triticum monococcum, Triticum dicoccoides (wild emmer), Aegilops tauschii, and Aegilops speltoides were found to have substantially higher levels of these minerals (two- to three-fold) in their grain than modern wheat cultivars (Rawat et al., 2009; Xu et al., 2011; Velu et al., 2014). Compared with the alternative durum allele, recombinant chromosome substitution lines (RSLs) with T. dicoccoides carrying the Gpc-B1 allele had a 12, 18, and 38% higher concentration of Zn, Fe, and protein content, respectively (Cakmak et al., 2004). High concentrations of Fe, Mn, and Zn in grain were stable across sites (Distelfeld et al., 2007). Hence, T. dicoccoides seems to be an interesting source for enhancing both protein and essential mineral content and concentration in wheat cultigens. Similarly, wild accessions of rice such as Oryza rufipogon, Oryza nivara, Oryza latifolia, and Oryza officinalis seem to be assets in rice improvement, showing higher values for Fe and Zn content than cross-bred cultivars (Banerjee et al., 2010; Anuradha et al., 2012). ### IDENTIFICATION OF MOLECULAR MARKERS FOR GRAIN FE AND ZN BIOFORTIFICATION IN CEREALS The finding of quantitative trait loci (QTLs) led to dissection of complex multigenic traits that were difficult to improve through crossbreeding before the progress made in DNA-aided analysis. QTL mapping for mineral nutrients in cereal grains has allowed the identification of many QTLs for both Fe and Zn (Table 2). Most of these QTLs, with a few exceptions, do not seem to be stable across sites. Furthermore, QTL mapping has also clearly indicated the role of epistasis in expression of these traits in cereal grains through interactions with other loci (Table 2). Unfortunately, there is no literature indicating so far a success story for marker-aided selection (MAS) for improving Fe and Zn in cereal grains, but some progress has been made that has laid the foundation stone toward breeding for Fe and Zn biofortification in cereals using MAS. For instance, some of the QTLs identified for Fe and Zn are co-localized, thereby suggesting common mechanisms for their transport. Furthermore, some QTLs for these mineral nutrients are also co-localized with those for other mineral elements such as phosphorus (P) and calcium (Ca), or other agronomically important traits including grain protein content and grain weight (Table 2). Fine mapping of candidate genes related to various QTLs could be a further step for developing biofortified germplasm. Rice is a model plant for cereal genetics. Chromosome 11 of rice bears a QTL for Zn concentration in the grain, which seems to be associated with OsNAC5—a transcription factor that appears to be related with the remobilization of Zn from green tissues to the seed (Lu et al., 2008; Sperotto et al., 2009, 2010). In unpolished rice grains, 10 candidate genes known for Fe and Zn homeostasis were localized in the QTL regions whereas another six candidate genes were close to QTLs on chromosomes 3, 5, and 7, respectively (Anuradha et al., 2012). Based on these results, Anuradha et al. (2012) emphasized the importance of candidate genes OsYSL1 and OsMTP1 for Fe; OsARD2, OsIRT1, OsNAS1, and OsNAS2 for Zn; and OsNAS3, OsNRAMP1, heavy metal ion transport, and APRT for both Fe and Zn biofortification of grain in rice. Recently, Norton et al. (2014) also found several QTLs for grain Zn and other elements in diverse rice genotypes using genome-wide association mapping, but the known Zn-related genes were not found in these regions, thereby showing the novelty of their results. The first QTL for grain Fe and Zn in wheat was found by Joppa et al. (1997), who mapped a major QTL (Gpc-B1) for grain protein content to chromosome 6BS in a population of recombinant inbred lines (RILs) that derived after crossing "Langdon" (LDN)—a durum wheat cultivar—and DIC6B—a chromosome substitution LDN line including wild emmer wheat. Subsequently, the Gpc-B1 locus was also found to be related to high concentrations of both Fe and Zn, as well as with fast leaf senescence. The dissection of the Gpc-B1 locus by positional cloning revealed that the gene underlying the Gpc-B1 locus encodes NAM1, which is a NAC transcription factor that belongs to a protein group that includes "No Apical Meristem" (NAM) in Arabidopsis thaliana (Uauy et al., 2006; Distelfeld et al., 2007). The ancestral wild wheat allele NAM-BI leads to fast senescence and enhances the remobilization of nutrients from the leaves to the developing grains. Modern wheat cultivars have instead a non-functional NAM-BI. Both Fe and Zn can be manipulated together because of the co-localization of their QTLs (Shi et al., 2008), whose mapping was facilitated by using RILs or diverse double-haploid (DH) populations (Table 2). The identification and tagging of DNA markers related to both traits provides an aid for crossbreeding, thereby accelerating biofortification for Fe and Zn in grains of cereals. TABLE 2 \| Main effect and epistatic quantitative trait loci (QTLs) associated with Fe and Zn accumulation in different tissues and their co-localization with other traits in cereal crops reported by different groups. (Continued) ### TABLE 2 \| Continued MQTL, main effect QTL; EQTL, epistatic QTL; GFe, grain iron; GZn, grain zinc; GPhy, grain phytic acid; LFe, leaf Fe; LZn, leaf Zn; Zneffi, Zn efficiency; ShZn, shoot Zn; ClZn, clum Zn; FlZn, flag leaf Zn; YlFe, young leaf Fe; YlZn, young leaf Zn; CobF, cob Fe; CobZn, cob Zn; GbioFe, grain bioavailable Fe; DH, double haploid; IL, inbred line; RIL, recombinant inbred line; Fn, segregating offspring. ### BREEDING FOR ENHANCEMENT OF FE AND ZN BIOAVAILABILITY: ROLE OF INHIBITORS AND PROMOTERS The ultimate goal of the breeding for Fe and Zn biofortification in cereals is to satisfy the requirement of the human body for these minerals. Thus, the bioavailability of these minerals should be measured according to the cereal-based foods consumed rather than as their quantity in the cereal grains. Considering the low bioavailability of these minerals, it seems to be difficult to meet this demand alone by enhancing the grain Fe and Zn content in cereals. Hence, Fe and Zn should be easily absorbable in the intestines—a difficult task due to inhibitors (e.g., phytic acid) or promoters such as prebiotics enhancing their absorption in the gut—to ensure their effective availability from cereal-based diets (Roberfroid, 2007; White and Broadley, 2009; Dwivedi et al., 2014). Phytic acid is an effective chelator of positively charged elements such as Ca, Fe, Mn, magnesium (Mg), potassium (K), and Zn, which after human or animal consumption binds to these minerals in the intestines forming mixed salts that are further excreted, thus resulting in mineral deficiency in human populations (Ali et al., 2010). Although phytate is considered an inhibitor of Fe and Zn bioavailability and therefore referred to as an anti-nutritional trait in cereal grains, it may have some health benefits such as being an antioxidant or anticarcinogen (Schlemmer et al., 2009). Furthermore, the important role of phytic acid has also been noted in plant traits such as seedling vigor or protection of seeds against oxidative stress during their lifespan (Doria et al., 2009). Hence, the existence of a minimum concentration of phytic acid in the cereal grains is still under scientific debate from health as well as crop performance perspectives. Nonetheless, various low-phytic acid (lpa) mutants have been found in barley, maize, rice, and wheat exhibiting 50 to 95% reduced phytic acid P (Rasmussen and Hatzack, 1998; Raboy et al., 2000; Pilu et al., 2003; Shi et al., 2003; Guttieri et al., 2004; Liu et al., 2007). However, the pleiotropic effects of these lpa mutations resulted in significant grain yield loss and also affected other agronomic traits such as poor seed germination along with low grain weight and starch accumulation, and poor plumpness, among other characteristics (Raboy et al., 2000; Pilu et al., 2003; Guttieri et al., 2006; Zhao et al., 2008). Thus, seeking available variability will assist in finding new genetic mechanisms that reduce phytate and avoid any grain yield penalty in cereals. About two-fold variation for seed phytate concentration has been observed in wheat and rice (Liu et al., 2006; Stangoulis et al., 2007). Interestingly, two QTLs for seed phytate concentration have been identified so far in rice; one each on chromosomes 5 and 12 accounting for phenotypic variance of 24 and 15%, respectively (Stangoulis et al., 2007). Genetic markers nearby these QTLs should be used for testing their efficacy as aids for selecting low-phytate lines. With the growing awareness about diet-related health problems, the presence of health-promoting natural compounds in staple foods, which was earlier considered of minor importance, has attracted greater attention in the food industry. Prebiotics are a group of carbohydrates that are known to confer benefits for human health by selectively promoting the growth or activity of gut microbiota (Dwivedi et al., 2014). Thus, prebiotics in cereal grains should be taken into account for enhancing Fe and Zn bioavailability while undertaking biofortification. To date, scarce research has reported the influence of prebiotics on the absorption of these mineral nutrients in humans and the prevalence of the natural variation and inheritance of these compounds in cereal grains. There is, however, significant genetic variability for inulin concentration in the grains of maize and rice, both of which have lower inulin concentration that those of rye and wheat (Genc et al., 2005; Huynh et al., 2008a). Similarly, substantial genetic variation has also been reported in grain fructan content ranging from 0.7 to 2.9, 3.6 to 6.4, and 0.9 to 4.2% of grain dry weight in the different genotypic lines and cultivars of wheat, rye, and barley, respectively (Boskov-Hansen et al., 2003; Huynh et al., 2008a; Nemeth et al., 2014). There is a relatively high level of low molecular weight soluble dietary fiber in wheat. It includes fructan, which was found in a double mutant sweet wheat (SW) line; however, seeds were severely shrunken and shriveled, and had reduced kernel weight (Shimbata et al., 2011). Nevertheless, the SW mutant can be utilized in breeding programs as a novel source to raise grain fructan levels. Among cereal crops, genetic mapping studies have been mainly performed in wheat for concentrations of grain prebiotics such as fructan, inulin, and arabinoxylan (Huynh et al., 2008b; Falcon, 2011; Nguyen et al., 2011). A total of five, four, and two QTLs explaining 2–27, 3–19, and 15–20% of phenotypic variation were detected in wheat for grain fructan, inulin, and arabinoxylan concentrations, respectively. Some epistatic QTLs were additionally detected for grain fructan and arabinoxylan concentration, although, their contributions were limited (Huynh et al., 2008b; Nguyen et al., 2011). Despite this, two QTLs each for fructan (6D and 7A), inulin (2BL.2 and 5BS), and arabinoxylan content (2A.1 and 4D.1) were major QTLs (PVE > 10%), suggesting molecular breeding to improve prebiotics significantly in grains of wheat. Recently, Huynh et al. (2012) mapped the fructan biosynthetic pathway gene coding for the enzyme sucrose:sucrose-1-fructosyltransferase (1-SST), which corresponds to the position of a major QTL on wheat chromosome 7A that affects the accumulation of grain fructan (Huynh et al., 2008b). Thus, identification of candidate genes underlying these QTLs would provide a basis for functional analysis and for the development of DNA markers that may assist molecular breeding with the aim of increasing prebiotic concentrations in the grain. ### BREEDING FOR HARMONY BETWEEN QUALITY AND SAFETY OF CEREAL GRAIN Besides food quality, food safety is also a "hot" topic that encourages scientists to engage in research related to health risks after consuming non-essential metals such as Cd and lead (Pb), and/or metalloids (arsenic, As), which have no beneficial role in plants, animals, or humans (Khan et al., 2015). Among these non-essential heavy metals, Cd particularly is known as highly phytotoxic, having a very low toxicity threshold level, and as a carcinogen, which is a great threat to human health. Nearly 27% of dietary Cd exposure is contributed by grain or grain products (Guttieri et al., 2015). Similarly, arsenic is also carcinogenic and can pose a serious threat to human health even at low concentrations. Moreover, the presence of high concentrations of these non-essential elements in cereal straw is still menacing because cereal straw is mainly used as livestock feed and thus these toxic elements may enter into the human food chain via contaminated meat or milk. Soil is a natural source of heavy metals, and their elevated concentration in soil can occur either naturally or through anthropogenic activities such as urban and industrial activities as well as from agricultural practices. These toxic metals contamination is a non-reversible accumulation process due to their long estimated half-life in soil. Thus, accumulation of toxic metals in cereal grains impacts significantly on nutritional quality and crop safety. Generally, metals commonly enter plants as divalent cations. It has been reported that increasing accumulation of Fe and Zn in seeds leads to a higher accumulation of Cd, which chemically resembles Fe and Zn. Thus, uptake of Cd in roots and then translocation to seeds appears to occur inside plants along nutrient translocation pathways (Krämer, 2009). The first overlapping QTLs for essential and non-essential metals were identified in the Zn/Cd hyperaccumulator Arabidopsis halleri, and the candidate gene underlying the major QTL was identified as AbHMA4 (Heavy Metal ATPase 4) (Hanikenne et al., 2008). Subsequently, HMA2 was determined to contribute to Cd and Zn translocation in rice (Clemens et al., 2013). The concentrations of essential mineral nutrients and nonessential metals in grains appear to be independently regulated because some independent grain Cd accumulation loci have been reported in cereals, such as the Cdu1 locus on 5BL in durum wheat (Knox et al., 2009) and one major QTL on 5AL in bread wheat (Guttieri et al., 2015). The identification of causal genes underlying these QTLs will provide more biological insights into Cd accumulation in cereal grains. Similarly, rice genotypes having dysfunctional OsNRAMP5 (Ishikawa et al., 2012) showed a substantial decrease in Cd uptake by roots, as well as Cd content in the straw and grain, but without decreasing the uptake of Fe by the roots, shoots, and straw (Ishimaru et al., 2012; Sasaki et al., 2012). These results suggest that a low grain Cd cereal cultivar can be developed without reducing the concentration of essential mineral nutrients through marker-aided breeding (MAB). Recent research has emphasized the importance of wild relatives for breeding high grain Fe and Zn in cereals crops. Nonetheless, possible pleiotropic effects of the introgression of elevated mineral nutrients need to be investigated by ICP–MS, thereby facilitating joint selection. ### OUTLOOK Globally, the committed efforts by CGIAR HarvestPlus have led to the integration of essential micronutrients as a core activity in the breeding programs of almost all major cereal crops. Considering the complex genetic mechanism of Fe and Zn accumulation in cereal grains, eradication of these mineral nutrient deficiencies by increasing their levels in cereal grains through conventional breeding is simply too difficult. In the post-genomic and computational systems biology era, the combination of high-throughput genomics and robust statistical analysis, particularly QTL mapping studies, has helped to dissect the molecular basis of natural diversity for complex quantitative traits in a better way. Recent molecular mapping studies clearly indicate the co-localization of QTLs for Fe and Zn with those for other potentially toxic metals such as Cd, Pb, and As. Available knowledge can be used to design targeted crosses for MAB targeting cereal cultivars with high levels of Fe and Zn. Undoubtedly, QTLs detected only for Fe or Zn have also revealed that plants may be able to differentiate between ### REFERENCES nutrients and chemically similar toxin ions. Although no information is available so far about the enhancement of toxic metals in cereal grains through cross breeding, there is fear of inadvertent breeding for these non-essential metals that are toxic to both plants and animals even in low concentrations. Moreover, modifications in the accumulation of these toxic elements that are of concern for food safety are rarely determined during research on mineral nutrients. Thus, utilization of natural genetic variation for these mineral nutrients through a molecular breeding approach seems to be more attractive in the future. Furthermore, existence of substantial genetic variability for Fe and Zn bioavailability inhibitors and promoters also offers good opportunities to increase the bioavailable forms of these mineral nutrients in cereal grains. Genes accounting for this variability have rarely, however, been found and, therefore, are not yet being used in breeding; however, this also seems to be a promising approach for the near future. Hence, Fe and Zn bioavailability from cereal grains may be improved through breeding by accumulating either anti-nutrient agents or prebiotics. Furthermore, both functional and genetic evidence along with genome sequencing will provide means for gaining more insights regarding the emerging biofortification genomics. ### AUTHOR CONTRIBUTIONS AG-O conducted the literature survey and together with SC wrote the first draft. RO edited and together with AG-O, SC, MG, and AM improved the manuscript writing. All authors read and approved the final manuscript. enhancing iron and zinc content in wheat. Genet. Resour. Crop Evol. 56, 53–64. doi: 10.1007/s10722-008-9344-8 zinc concentration in diploid A genome wheat. J. Hered. 100, 771–776. doi: 10.1093/jhered/esp030 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Garcia-Oliveira, Chander, Ortiz, Menkir and Gedil. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Applications of New Breeding Technologies for Potato Improvement Amir Hameed1†, Syed Shan-e-Ali Zaidi <sup>2</sup> \* † , Sara Shakir 2† and Shahid Mansoor <sup>2</sup> \* <sup>1</sup> Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan, <sup>2</sup> Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan ### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil ### Reviewed by: Sunette M. Laurie, Agricultural Research Council of South Africa (ARC-SA), South Africa Felipe Dos Santos Maraschin, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil #### \Correspondence: Syed Shan-e-Ali Zaidi [email protected] Shahid Mansoor [email protected] ### †Present Address: Amir Hameed, Akhuwat-Faisalabad Institute of Research, Science and Technology, Faisalabad, Pakistan Syed Shan-e-Ali Zaidi, Gembloux Agro-Bio Tech, University of Liège, Gembloux, Belgium Sara Shakir, Boyce Thompson Institute, Ithaca, NY, United States ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 01 December 2017 Accepted: 11 June 2018 Published: 29 June 2018 ### Citation: Hameed A, Zaidi SS, Shakir S and Mansoor S (2018) Applications of New Breeding Technologies for Potato Improvement. Front. Plant Sci. 9:925. doi: 10.3389/fpls.2018.00925 The first decade of genetic engineering primarily focused on quantitative crop improvement. With the advances in technology, the focus of agricultural biotechnology has shifted toward both quantitative and qualitative crop improvement, to deal with the challenges of food security and nutrition. Potato (Solanum tuberosum L.) is a solanaceous food crop having potential to feed the populating world. It can provide more carbohydrates, proteins, minerals, and vitamins per unit area of land as compared to other potential food crops, and is the major staple food in many developing countries. These aspects have driven the scientific attention to engineer potato for nutrition improvement, keeping the yield unaffected. Several studies have shown the improved nutritional value of potato tubers, for example by enhancing Amaranth Albumin-1 seed protein content, vitamin C content, β-carotene level, triacylglycerol, tuber methionine content, and amylose content, etc. Removal of anti-nutritional compounds like steroidal glycoalkaloids, acrylamide and food toxins is another research priority for scientists and breeders to improve potato tuber quality. Trait improvement using genetic engineering mostly involved the generation of transgenic products. The commercialization of these engineered products has been a challenge due to consumer preference and regulatory/ethical restrictions. In this context, new breeding technolgies like TALEN (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated 9) have been employed to generate transgene-free products in a more precise, prompt and effective way. Moreover, the availability of potato genome sequence and efficient potato transformation systems have remarkably facilitated potato genetic engineering. Here we summarize the potato trait improvement and potential application of new breeding technologies (NBTs) to genetically improve the overall agronomic profile of potato. Keywords: CRISPR, genome editing, nutritional quality, potato, TALEN ## INTRODUCTION The rising food demand in a populating world will require a proportional increase in the food source. In contrary, several factors like climatic change, industrialization, and urbanization have overburdened the existing agriculture lands and food resources (Badami and Ramankutty, 2015). Other factors causing food decline include various biotic and abiotic stresses continuously affecting 102* crops worldwide. With the technological advancements and joint public-private partnership, several crops with enhanced nutritional profile have been developed using the existing gene pool (Ricroch and Henard-Damave, 2016; Ma X. et al., 2017). Potato (Solanum tuberosum L.), a solanaceous food crop, is ranked fourth among the major staple crops after maize, rice, and wheat. It can provide more carbohydrates, proteins, minerals, and vitamins per unit area of land and time as compared to other potential food crops (Zaheer and Akhtar, 2016). In addition to being a raw marketable product, potato is largely used in industry for making processed food products, alcohol, starch, animal feed and for biofuel production (Scott and Suarez, 2012; Liang and McDonald, 2014). Short crop duration and wide climatic adaptability have facilitated potato to spread across diverse geographical borders from its South American origin. Today, more than three thousand potato cultivars are widely distributed in more than 125 countries, particularly under temperate, subtropical and tropical regions covering a major economic share in the global agricultural market (Birch et al., 2012). For the last two decades, potato cultivation and utilization have also been notably increased in developing countries such as Bangladesh, India, and China (Zaheer and Akhtar, 2016). In terms of nutrition, potato is a complex source of nutrients (vitamins, carotenoids, anti-oxidant phenolics, proteins, magnesium etc.), and some anti-nutrients (primarily glycoalkaloids). On average, potato tubers contain 77% water, 20% carbohydrates, and less than 3% of proteins, dietary fiber, minerals, vitamins and other compounds (Zaheer and Akhtar, 2016). Comprehensive information regarding the tuber composition of different potato cultivars is described by Burlingame et al. (2009). In low-income food-deficit countries (http://www.fao.org/countryprofiles/lifdc/en/), potato could replace other high-priced foods and can be sustainably used as a cheap food giving enough calories (93 kcal/100 g tuber) to sustain a normal life (Burlingame et al., 2009). The global importance of potato is unquestionable and to commercialize its role in defeating food-shortage, poverty, and predominantly malnutrition, United Nations celebrated 2008 as the "International Year of the Potato" (http://www.fao.org/ potato-2008/en/). Several breeding and molecular approaches have been employed for trait improvement in potato. Conventional breeding techniques for potato improvement are directed to increase yield, processing, and storage-quality (Halterman et al., 2016). Potato breeders incorporated resistance against early and late blight disease by crossing hybrid lines with wild species (S. brevidens and S. bulbocastanum) which inherited resistance against fungal pathogens (Naess et al., 2000; Tek et al., 2004). Although conventional breeding has been successfully employed for targeted trait improvement with less intraspecific variability, the progress is relatively slow and limited due to the phenotypic characterization of leading individuals in successive generations. In addition, the search of useful genetic variability in wild relatives could be laborious and its introgression in cultivated variety can be another challenging task. High heterozygosity and tetraploid nature of the potato genome (Consortium, 2011) are major drawbacks in breeding efforts to improve potato because of allelic suppression at each breeding cross (Lindhout et al., 2011). Other factors may include intra-species incompatibilities and inbreeding depression that causes failure in trait incorporations in polyploid crops through conventional breeding. In this context, new breeding technologies (NBTs) offer a leading hand for trait improvement in crop plants and provide a platform for precise and robust plant genome editing. These NBTs include, but are not limited to, the cutting-edge genome editing approaches like clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) (Jinek et al., 2012; Schaart et al., 2016; Weeks et al., 2016). Although developed recently, CRISPR system has been effectively employed for trait improvement of several economically important crops like wheat, maize, rice, cassava, cotton, soybean, and potato (Puchta, 2017). The introduced traits include herbicide tolerance, fungal/bacterial/viral disease resistance, drought tolerance, and increased shelf life, leading to overall improved quality and production. The working methodology and the anticipated role of these NBTs in plant genetic engineering have been extensively reviewed (Bortesi and Fischer, 2015; Mahfouz et al., 2016; Schiml and Puchta, 2016; Puchta, 2017; Weeks, 2017; Zaidi et al., 2017a,b, 2018). The current review provides a comprehensive information on different genetic approaches, including NBTs, that have been successfully employed to enhance the nutritional value of potato (Tables 1, 2). Moreover, we summarize the data on transgenic potato commercialized so far (Table 3) and the major concerns associated with their regulatory approvals. ### CONSTRAINS TO POTATO PRODUCTIVITY AND QUALITY The sustainable potato production faces a number of challenges due to biotic stresses (viruses, bacteria, fungal, insect pests) and abiotic stresses (drought, salinity, temperature, frost and postharvest problems, i.e., accumulation of reducing sugars during cold storage). ### Diseases and Insect Pests Affecting Potato Most of the potato diseases are due to the diverse prevalence of phytopathogens of which viruses are of prime importance. Cultivated potato is susceptible to around 40 different viral and 2 viroid species (Salazar, 1996). Among the dominating viruses, Potato virus Y (PVY, genus; potyvirus), Potato leafroll virus (PLRV, genus; polerovirus), and Potato virus X (PVX, genus; potexvirus) are probably the most diverse and devastating viruses infecting potato worldwide (Fletcher, 2012; Hameed et al., 2014; Steinger et al., 2014). Viral diseases appear as necrotic strains on leaves/tubers, mosaic, and overall stunted growth to plant, leading to reduced yield and poor-quality tubers. Moreover, several bacterial diseases (soft rot/blackleg caused by Dickeya solani, common scab caused by Streptomyces scabies) (Buttimer et al., 2017), and fungal diseases (late blight caused by Phytophthora infestans, powdery scab caused by Spongospora subterranea; Arora et al., 2014; Balendres et al., 2016) are Transgenesis: Introducing an exogenous gene "transgene" into a living organism so that the organism will stably exhibit a new property and transmit that property to next generation. also severely deteriorating potato quality worldwide. Late blight affected potato plants exhibit water-soaked leaves having necrotic lesions and irregular colored tissue in tubers making them hard, dry and more susceptible to other microbial diseases. Virus resistance in potato has been engineered through different approaches ranging from simple plant breeding to advanced genetic engineering. Transgenic approaches to engineer virus resistance in potato are seemed to be more appropriate than conventional breeding due to its polyploid nature making difficulties for the introgression of resistance genes. Thus, RNA interference (RNAi)- mediated resistance targeting viral coat protein (CP) region has been demonstrated in potato, where single or multiple RNA viruses have been targeted with different success levels; such as PVY- resistance (Missiou et al., 2004); PVY, and PLRV-resistance (Chung et al., 2013); and PVX, PVY, and Potato virus S (PVS)-resistance (Hameed et al., 2017). The current scenario of GM potatoes being commercialized in some countries encompasses viral resistant potatoes generated through genetic engineering (Mathur et al., 2017). Potato pests cause direct damage to potato crop in the forms of necrosis, deformations of plant tissues and/or indirect damage by facilitating the pathogen dispersal, especially for viruses. Important destructive insects affecting potato include Colorado potato beetle (Leptinotarsa decemlineata) (Casagrande, 2014), peach-potato aphid (Myzus persicae) (Bass et al., 2014), potato tuber moth (Phthorimaea operculella) (Liu et al., 2018) etc. The extensive use of chemical insecticides on insect pests has led to the evolution of insecticide-resistance in particular insects, thus posing alarming threats. To effectively control their incidence in planta, genetic engineering has offered some promising solutions like introgression of insecticidal proteins/toxins (Palma et al., 2014), RNAi-mediated insect resistance (Zhang J. et al., 2017) and CRISPR-Cas9-mediated crop protection (Douglas, 2017) etc. In TABLE 2 \| Applications of some new breeding technologies for potato trait improvement. potato, RNAi technology was used to engineer resistance against Colorado potato beetle (Zhang et al., 2015). Sap transmitted RNAi reagents (long double-stranded (ds) RNAs in chloroplasts) resulted in more than 80% of the reduced expression of insect targeted genes (β-actin gene) and triggered a lethal RNAi response destructive to its larvae (Zhang et al., 2015). ### Agronomic Attributes Affecting Potato Farming systems comprising of different agronomic attributes like tillage, nutrient management, and crop rotation significantly affect potato tuber productivity and quality. Due to its shallow root-system, potato needs a fair supply of nutrient inputs to maintain its tuber vigor and yield (Alva et al., 2011). Research has shown the influence of different farming practices on tuber quality parameters like tuber dry mass accumulation, enhanced nutrient/mineral concentration and yield improvements (Brazinskiene et al., 2014; Tein et al., 2014; Nyiraneza et al., 2015). Through adopting a potato-legume crop rotation, (Qin et al., 2017) observed a positive influence on soil microbiota coupled with significantly improved tuber yield up to 19% when compared with the continuous cultivation of potato crop only. Integrated crop rotations with an exogenous supply of organic and mineral [nitrogen (N), phosphorous (P), and potassium (K)] fertilizers significantly influenced potato tuber N, nitrate, magnesium (Mg) and P concentrations when compared with non-fertilized controls (Tein et al., 2014). Leonel et al. (2017) analyzed five potato cultivars for their tuber chemical composition in response to different concentrations of available P supplemented with uniform cultural practices. Potato tubers fertilized with increased P exhibited a significant positive influence of tuber dry matter and protein/starch contents and a lower concentration of total sugar contents (Leonel et al., 2017). The chemical composition of potato tubers is a prerequisite for determining the nutritional and processing quality of industrial perspectives. Understanding the importance of organic products, Lombardo et al. (2017) evaluated the nutritional value of organic vs. conventionally grown potatoes. Field trials of yellow-fleshed potato cultivars growing under organic cultivation produced high-quality tubers having enhanced concentrations of phenolics, reduced nitrate and a more attractive tuber flesh color (Leonel et al., 2017). ### Climatic and Soil Factors Affecting Potator A number of abiotic stresses ranging from soil to climate significantly affect the potato productivity and quality during its growth and/or after harvest. Potato cultivation performs better under cool condition (19◦C) and is vulnerable to high temperatures (Kim et al., 2017). A fairly low temperature promotes the first tuber set and sudden elevations in temperature during this early tuberization significantly affect tuber yield and size (Zhou et al., 2017). A drastic reduce in potato tuber yield ranging from 3 to 11% per 1◦C rise in temperature was observed across various geographical locations (Fleisher et al., 2017; Kim et al., 2017). During the early growth stage of potato, low freezing and/or frost attacks severely damage the young plantlets and could reduce the tuber yield and quality (Chang et al., 2014). Drought and salinity are other important abiotic stresses having adverse effects on potato cultivation. Potato cultivars respond differentially to drought conditions and mostly exhibit various physiological and morphological changes in tuberization \Unknown. R&D, Research and Development; EPA, U.S. Environmental Protection Agency; FDA, Food and Drug Administration; EC, European Commission; EU, Europe; CPB, Colorado Potato Beetle; PLRV, Potato leafroll virus; Cry3A and Cry2a1, Bacillus thuringiensis genes; VInv, Vacuolar acid invertase; Asn1, Asparagine synthetase-1 gene; Rpi-vnt1, Late Blight resistance gene from wild potato (Solanum venturii); AmAI, Amaranthus hypochondriacus1; GDP, Arabidopsis thaliana <sup>L</sup>-galactose phosphorylase; GBBS, Granule bound starch synthase; BBSRC, Biotechnology and Biological Sciences Research Council; WUR, Wageningen University and Research Centre; NIPGR, National Institute of Plant Genome Research. and plant growth (Chang et al., 2018). The major impact of this water stress has been recorded in the form of reduced tuber yield due to a loss of internal water pressure during tuber bulking and maturation (Stark et al., 2013). Salinity, another acute abiotic stress causes many inhibitory effects on plant growth and development (Parihar et al., 2015). Salinity stress in potato severally affects its productivity by causing enhanced oxidative stress, reduced photosynthesis and significantly reduced tuber yield. Research efforts through interspecific breeding in potato resulted in improved tolerance to salinity and oxidative stresses (Jbir-Koubaa et al., 2015). ### Post-harvest Factors Affecting Potato The post-harvest storage of potato tubers is another criterion for determining their end-product processing quality. Usually, this long-term storage is accompanied with various storage diseases like soft rot, black dot, and Fusarium dry rot which significantly reduces the tuber quality and unfit it for further processing (Usall et al., 2016). Soil-transmitted black dot, caused by Colletotrichum coccodes imparts brown necrotic lesions/stains on tuber skin and promotes their rapid decay (Brierley et al., 2015). Storage temperature and durations are two important factors determining the tuber susceptibility to black dot disease and could be managed to prevent the quality losses in potato (Peters et al., 2016). The tuber harvesting date also influences tuber quality during the long-term storage. Makani et al. (2015) observed the storage quality of potato cultivars in response to harvest time and subsequent storage. The results showed that the tubers harvested at full maturity retained their quality during storage in contrary to early harvested tubers (less mature), which exhibited a significant loss in tuber dry matter and ascorbic acid contents (Makani et al., 2015). Potato dormancy characteristics are other challenging factor determining the tuber quality during storage. Dormancy is an innate ability to sustain sprouting for a time and after its natural breakage, sprouting starts which cause various quality issues. Dormancy could be regulated in potato tubers through the topical applications of phytohormones, such as ethylene which has the ability to suppress bud formation/sprouting (Sonnewald and Sonnewald, 2014). Potatoes are usually subjected to cold-storage (4–8◦C) in order to ensure a continuous supply to consumers/markets throughout the year. This cold-storage is accompanied by elevated levels of reducing sugars in the tuber, a phenomenon termed as "Cold-Induced Sweetening: CIS" (Bhaskar et al., 2010). During CIS, tuber starch content is biochemically converted to sugars (sucrose) through the cohesive activity of several hydrolytic enzymes (Sowokinos, 2001). The elevated sucrose is subsequently transported inside a vacuole where it is further reduced to glucose and fructose through the activity of a host gene (vacuolar acid invertase, VInv) (Sowokinos, 2001; Bhaskar et al., 2010). The CIS affected tubers when used as feedstock for high-temperature processing gives rise to the accumulation of a dark brown, bitter tasting product, i.e., acrylamide. The rising acrylamide contents in food products is a huge concern to global food safety as well as to end-chain consumers (Vinci et al., 2012). Several reports depict the alarming levels (up to 70%) of acrylamide in food products that mainly come through the intake of fries, chips and other fried potato products (Pedreschi et al., 2014; McCombie et al., 2016; Esposito et al., 2017). ### ENHANCING NUTRIENT CONTENTS IN POTATO For the last two decades, several efforts have been conducted to improve the nutritional traits of potato. The following section describes the information regarding nutrient enhancement in potato and is summarized in Table 1. ### Increased Protein Content The risk of protein deficiency is more in the countries where people take protein-deficit diet as a staple food (Chakraborty et al., 2010). Unfortunately, the cultivated potato contains fewer proteins (0.85–4.2%) lacking lysine, tyrosine, and some other essential amino acids (Burlingame et al., 2009). To deal with this limitation, scientists have engineered potato with enhanced protein content through constitutive expression of tuber-specific gene, Amaranthus hypochondriacus1 (AmA1) (Chakraborty et al., 2000, 2010). The AmA1 gene encodes for a seed protein, albumin: a non-allergic protein containing essential amino acids and considered safe for human/animal consumption [safety accredited by the World Health Organization (WHO)]. In transgenic potato, the enhanced protein (albumin) localizes inside cytoplasm/vacuole. The tubers of seven engineered potato cultivars showed an increased protein content up to 60% as compared to controls (Chakraborty et al., 2010). In addition to increased protein content, the transgenic potato also showed an accelerated rate of photosynthesis that ultimately increased the total biomass/yield of plants. Recently, methionine content (an essential amino acid involved in multiple cellular pathways) was significantly increased in transgenic potato cultivar (cv.) Desirée (Kumar and Jander, 2017). By using RNAi technology, overexpression of an exogenous gene Arabidopsis thaliana cystathionine γ-synthase (AtCGS), along with the suppression of a host gene S. tuberosum methionine γ-lyase (StMGL), resulted in nearly a double concentration of free methionine inside transgenic tubers as compared to control tubers (Kumar and Jander, 2017). Moreover, the experimental studies of engineered plants showed no morphological and yield differences when compared with control plants. Other studies were also conducted to increase the protein content in potato but met with limited success and yield penalties (Zeh et al., 2001; Dancs et al., 2008; Rinder et al., 2008; Galili and Amir, 2013). ### Increased Vitamin and Carotenoid Contents Several studies have been made to increase the vitamin content in potato, for example, expressing an exogenous gene, A. thaliana <sup>L</sup>-galactose phosphorylase (GDP) showed a 3-fold increase in ascorbate contents (vitamin C) (Bulley et al., 2012). Carotenoids are phytonutritive, anti-oxidative, lipophilic compounds (precursors to vitamin) present in many fruits and vegetables (Dellapenna and Pogson, 2006), and provide nutritional benefits in terms of increased vitamin uptake. Introduction of cauliflower Orange (Or) gene has shown a net increase in carotenoid content (pro-vitamin A) in coldstored tubers (Li et al., 2012). Among other carotenoids (Lutein, zeaxanthin, violaxanthin, neoxanthin), β-carotene concentration is considerably low in potato (Ezekiel et al., 2013). RNAi approach was utilized to silence the β-carotene hydroxylase (bch) gene that showed a significant increase in β-carotene and lutein contents in the tubers (Van Eck et al., 2007). Another study reported a 20-fold increase in tuber carotenoid contents through expressing three bacterial genes involved in carotenoid biosynthesis (Diretto et al., 2007). Similarly, transgenic potato cv. Taedong Valley was produced, over-expressing GLOase gene (L-gulono-γ-lactone oxidase from rat cells) that showed an enhanced (141%) content of <sup>L</sup>-Ascorbic acid (vitamin C) (Upadhyaya et al., 2010). ### Increased Calcium Content Being nutritious with several other elements, the cultivated potato is a poor source of Ca (Weaver et al., 1999). To address this deficiency, Park et al. (2005) utilized a transgenic approach through expressing an exogenous gene, Arabidopsis H+/Ca2<sup>+</sup> transporter (sCAX1) in potato cv. Russet Norkotah. The regenerated plants expressing sCAX1 gene showed a significant increase (up to 3-fold) of Ca contents in tuber as compared to controls. Field trials and morphological data from three consecutive crop generations proved the stable integration of enhanced Ca trait with no alteration in tuber yield and other growth/morphological characters. Potato with enhanced Ca contents could be potentially used as a dietary source, more specifically in countries where potato is a staple food (Park et al., 2005). ### Increased Phenolic Contents In potato, 80% of the phenolic compounds are present in the form of caffeoyl quinic acids (CQAs) (Brown, 2005). Recently, Li et al. (2016) conducted a study to increase the CQAs content in potato tubers. Tuber specific constitutive expression of an exogenous gene, flavonol-specific transcriptional activator (AtMYB12: derived from A. thaliana) showed a significant increase (>3-folds) of CQAs and total flavonoid content. Importantly, they utilized a selectable marker-free approach to facilitate the downstream regulatory approvals (Daniell, 2002). The increased phenolic contents being imposing health benefits also induce some antimicrobial properties to plants, particularly with reduced fungal infections (Li et al., 2016). ### Increased Starch Contents Potato tubers are a rich source of dietary starch and can provide a significant calorie intake in food-deficit countries. Starch is primarily composed of two structural components, amylose, and amylopectin, which are biosynthesized through cohesive actions of several enzymes. Extensive studies have been conducted to improve the digestible amylose content in potato by engineering different steps of starch biosynthesis pathway (Schwall et al., 2000; Hofvander et al., 2004). Tuber specific RNA silencing of two host genes (SBEI, SBEII), involved in starch branching pathway, resulted in the generation of potato with enhanced amylose content (Andersson et al., 2006). Recently, amylose contents were significantly increased (28–59%) in non-genetically modified potatoes by introducing a recessive allele (gene marker: IAm) from wild potato (S. sandemanii) into cultivated potato (S. tuberosum) through marker-assisted crossing (Krunic et al., 2018). Plant-based oils are promising for the near future as a potential feedstock for a renewable energy. Currently, biofuel research is more focused on engineering crops with enhanced oil contents through genetic manipulation in lipids/triacylglycerol (TAG) synthesis pathways (Vigeolas et al., 2007; Vanhercke et al., 2014; Zale et al., 2016). Contrary to high starch content, oil (lipids) concentration is very low in potato tubers. Some recent studies have shown the enhanced TAG content in the engineered potato tubers (Hofvander et al., 2016; Liu et al., 2017). Tissuespecific constitutive expression of three genes (WRI1, DGAT1, and OLEOSIN) resulted in a 100-fold increase in TAG content in tuber as compared to controls (Liu et al., 2017). However, this TAG increase was also accompanied by a depletive nutritional effect in terms of significantly reduced starch (amylose) and accumulated sugar (sucrose) levels. Further exploration of this mechanism revealed a better understanding of negative impacts of TAG accumulation on tuber amylose and phosphate contents, as well as needs to optimize genetic engineering for particular traits (Mitchell et al., 2017). ### REDUCTION OF ANTI-NUTRIENT CONTENTS IN POTATO Another strategy to improve the nutritional quality of food is by reducing the anti-nutrient elements. There is no lethal toxicity reported with the consumption of potato as food (Zaheer and Akhtar, 2016), however, some anti-nutrient elements like steroidal glycoalkaloids (SGAs) (0.071–175 mg/100 g), primarily α-solanine, and α-choconine accumulate in tubers during crop maturation (Burlingame et al., 2009). These SGAs when present in higher amounts in food may cause neuro-toxic and/or nutrient absorption problems (Itkin et al., 2013). Biosynthesis of SGAs involves a concurrent expression of two key enzymes, uridine 5' diphosphate, and glycosyltransferase which biochemically react with cholesterol, sugars, and other nitrogenous compounds to build up the glycoalkaloid molecules (Itkin et al., 2011). RNAimediated silencing of the host gene, Glycoalkaloid metabolism 4 (GAME4) in potato showed a significant decrease (up to 74 fold) in SGAs content in leaves and tubers (Itkin et al., 2013). Importantly, many wild species of potato produce high levels of SGAs naturally (Gregory et al., 1981), therefore breeders must be careful to map the SGAs-gene (s) linkage with the desired traits when using the wild germplasm as a genetic resource. Usually, raw potato is processed (fried, baked, mashed, microwaved) into various food products like snaps, fries, chips, etc. prior to eating (Zaheer and Akhtar, 2016). Sometimes, potato processing results in hazardous compounds causing obesity, cardiovascular diseases, and/or neurotoxicity. The first report of acrylamide, a potential neurotoxin and carcinogenic element, presence in potato fried products raised a debate among food regulatory authorities and processing industry (Tareke et al., 2002). Since then, several studies were conducted to explore the acrylamide formation during "Maillard Reaction," a reaction among tuber asparagine contents, reducing sugars (primarily glucose and fructose) and free α-radicals present in cooking oil during high-temperature processing of potato (Stadler et al., 2002; Friedman, 2003; Vinci et al., 2012). Contrary to cultivated tetraploid potato (S. tuberosum), some diploid species of wild potato are naturally resistant to CIS. Through quantitative trait loci (QTL) mapping and other molecular studies, scientists have identified some recessive genes associated with CIS resistance in wild species. Potato breeders tried to incorporate these genes in cultivated potato in order to reduce the CIS effect but met with limited success. Therefore, different molecular strategies have been applied to reduce the formation of acrylamide through indirectly mediating the CIS mechanism at the cellular level (Bhaskar et al., 2010; Li et al., 2013; Zhu et al., 2016; Hameed et al., 2018). Tuberspecific constitutive expression of VInv gene in anti-sense binary constructs resulted in significant reduction of reducing sugar content in cold-stored tubers. High-temperature processing of food products derived from these transgenic lines showed an 8-fold decrease in acrylamide content as compared to controls (Ye et al., 2010). In another study, RNAi-mediated simultaneous silencing of potato asparagine synthetase genes (StAS1 and StAS2) and VInv gene significantly reduced the CIS process as well as asparagine content in transgenic potato cv. Russet Burbank (Zhu et al., 2016). Tubers derived from these CIS resistant transgenic lines showed a significant reduction (15 fold) of acrylamide content in fried potato products. The firstgeneration biotech potato (Simplot's Innate TM) was engineered to have lower reducing sugars levels and reduced asparagine contents to address the acrylamide forming problems during potato processing (Halterman et al., 2016). ### NEW BREEDING TECHNOLOGIES USED FOR INCREASING NUTRITIONAL QUALITY OF POTATO Gene pyramiding in polyploid crops using conventional breeding is a difficult, laborious, and time-consuming (Weeks, 2017). In potato, several breeding efforts have been made for particular trait improvement using wild species germplasm but met with limited success (Carputo and Barone, 2005). The presence of four copies (alleles) of genes in the tetraploid (2n = 4x = 48) genome of cultivated potato (S. tuberosum) makes it difficult for researchers/breeders to precisely edit the genome using conventional breeding tools (Consortium, 2011). Thus, NBTs such as CRISPR/Cas9, TALENs, and ZFNs offer great potential for expediting genome editing in a more precise and time-saving way (Mahfouz et al., 2016; Petolino et al., 2016; Schiml and Puchta, 2016). In case of potato, both TALENs (Sawai et al., 2014; Nicolia et al., 2015; Clasen et al., 2016), and CRISPR/Cas9 (Butler et al., 2015; Wang et al., 2015) technologies have been efficiently utilized for precise genome editing (Table 2). In 2014, the first attempt of utilizing TALENs technology in potato genome editing paved the way for the next technology shifts. Sawai et al. (2014) utilized TALENs approach in potato (S. tuberosum cv. Sassy) to silence a host gene, Sterol side chain reductase 2 (StSSR2) that is predominantly involved in cholesterol biosynthesis and a precursor to many toxic SGAs formations. Transgenic expression of TALENs constructs generated a site-specific mutation of variable size (nucleotide deletion/insertion) in four alleles of StSSR2 gene. The transgenic potato with knock-out StSSR2 activity showed a significant reduction of SGAs contents without affecting plant growth, thus eliminating an anti-nutritional factor (Sawai et al., 2014). In another study, protoplast delivery of TALENs constructs resulted in a significant mutation frequency (7–8%) at targeted gene loci, i.e., Acetolactate synthase (ALS) in transgenic potato cv. Desirée (Nicolia et al., 2015). Sequencing analysis of ALS-mutated lines confirmed the targeted protein disruption either through amino acid substitutions, truncations, and/or frameshift mutations and importantly mutated lines showed no phenotypic differences compared to controls (Nicolia et al., 2015). Wang et al. (2015) reported the use of CRISPR/Cas9 system for inducing efficient targeted mutagenesis in potato. Agrobacterium-mediated transformation of cells with CRISPR constructs resulted in efficient site-specific mutation in host gene, Auxin/indole-3-acetic acid (StIAA2) engineered for altered Aux/IAA protein expression (Wang et al., 2015). In a later study, Butler et al. (2015) targeted Acetolactate synthase1 (ALS1) gene in potato through the Agrobacterium-mediated delivery of CRISPR/Cas reagents. Importantly, they utilized both tetraploid (S. tuberosum cv. Desirée) and diploid (MSX914-10-X914-10) potato as explant for their experiments. Stable expression of CRISPR/Cas reagents resulted in site-specific mutations (ranged from 3 to 60%) in ALS alleles and were stably heritable (87–100%) in successive diploid and tetraploid potato generations (Butler et al., 2015). To improve the nutritional value of potato tubers, TALENs technology has been used to interrupt the VInv activity in order to reduce the accumulation of reducing sugars during cold-induced storage (CIS) (Clasen et al., 2016). Protoplastmediated transformation of potato cv. Ranger Russet with TALENs constructs resulted in knockout of VInv alleles in transformed plants (Clasen et al., 2016). Interestingly, 5 out of 18 transformed lines showed a nearly complete silencing of VInv gene having minimal or no detectable CIS activity. Furthermore, high-temperature processing (fried chips) of transgenic derived tubers resulted in light brown products having a significantly lowered level of dietary acrylamide. Importantly, in downstream characterization, few of transgenic lines showed a complete absence of TALENs sequences, thus offering a transgene-free approach. Other efforts to incorporate nutritional traits in potato include starch alterations using TALENs (Kusano et al., 2016) and CRISPR/Cas9 (Andersson et al., 2017). Through designing a novel delivery system, termed "Emerald–Gateway TALEN system," Kusano et al. (2016) targeted a host gene, Granule-bound starch synthase (GBSS) in potato for site-specific mutation. GBSS is predominantly involved in amylose biosynthesis during starch granulation. Its disruption may reduce the amylose content and amylose/amylopectin ratio (Zeeman et al., 2010), and thus might affect starch quality in potato tubers. Agrobacterium-mediated transformation of potato cells with TALEN constructs resulted in three types of stable mutations in regenerated lines, dominantly having a deletion mutation (63 nucleotides deletion) (Kusano et al., 2016). Recently, CRISPR/Cas9 technology has been used to efficiently silence the GBSS in potato cv. Kuras (Andersson et al., 2017). Protoplast transformation with CRISPR/Cas9 constructs resulted in a site-specific mutation in all (four) alleles of GBSS gene in 2% of transformed lines. Full knock-out of targeted genomic sites resulted in complete loss of the GBSS activity and yielded an altered starch quality in transgenic tubers when compared to controls (Andersson et al., 2017). Genomewide analyses coupled with transcriptomics, proteomics and metabolomics will further dissect the molecular basis of starchrelated traits of potato and could facilitate/accelerate the starch modifications by using NBTs. The production of high-quality starch in potato may be of current research interest to meet the demands of food and industrial sectors. In another study, Butler et al. (2016) utilized a geminivirus replicon (GVR) vector for delivering sequence-specific nucleases (SSNs) to target the potato herbicide tolerance gene ALS1 and regenerated transformants carrying a point mutation in ALS1 gene were confirmed for herbicide susceptibility. Forsyth et al. (2016) reported the targeted integration of transgene into a pre-selective, transcriptionally active site of potato genome using TALEN system coupled with a molecular marker, i.e., the mutated Acetolactate synthase (ALS) gene. Potato Ubi7 (constitutively expressing gene) was selected as a target for TALEN and after its functional confirmation in a transient system (N. benthamiana), Agrobacterium-mediated transformation was used to develop transgenic potatoes (S. tuberosum cv. Ranger Russet) (Forsyth et al., 2016). Importantly, the molecular confirmation of transgenic lines showed a single copy of transgene in most of the regenerated events. This could help in downstream transgenic characterization by reducing the workload of generating multiple independent lines for random transgene insertions. Their work established the efficacy of TALENs for achieving a more precise and site-specific genome editing in potato for trait incorporation. However, stable transformation of TALEN reagents carrying bacterial genes (TALE DNA binding domain from Xanthomonas) in plants may trigger the GM concerns having transgenes as codified by the regulatory authorities. Recently, transient expression of TALENs, delivered through non-viral Agrobacterium-mediated transformation, yielded targeted mutations in two potato cultivars, Russet Burbank and Shepody (Ma J. et al., 2017). The infiltrated TALEN constructs were meant to induce mutations in two different host genes, i.e., (i) 1,4-alpha-glucan branching enzyme (SBE1), (ii) Vacuolar invertase 2 gene (StvacINV2). The regenerated lines were confirmed for targeted chromosomal mutation through deep sequencing (Illumina), that revealed three types of induced mutations having dominantly deletion mutations in both of cultivars. TALEN technology through agroinfiltrations could be effectively used to induce targeted mutation for improving some elite potato cultivars (Ma J. et al., 2017). Figure 1* illustrates a schematic model of NBTs application for incorporating desired modifications in the potato genome to enhance nutritional improvements. ZFNs are first-generation genome editing nucleases engineered to make DSBs through fusions of artificial, sequencespecific zinc finger proteins with the nonspecific DNA cleavage domains of the FokI restriction endonuclease (Kim and Kim, 2014). The applications of ZFNs for genetic engineering has been limited to crops like tobacco (Cai et al., 2009), Arabidopsis (Zhang et al., 2010), and soybean (Curtin et al., 2011) and not in potato and other horticultural crops (Gaur et al., 2018). The limited examples of ZFNs-mediated genome modification in plants are might be due to some disadvantageous such as low success rate (∼24%), low or variable mutation rate (∼10%), high off-target effects, and technically difficult in designing feasibility (Xiong et al., 2015; Zhang H. et al., 2017). These challenges have greatly narrowed the spectrum of ZFNs technology for adoption by the scientific community. The studies discussed in this section provide comprehensive information regarding the utility of some NBTs for potato genome editing. Although, the applications of these tools are unlimited in the context of genetic engineering, the selection of suitable genomic targets and efficient editing tool is a critical prerequisite to get the desired goals. For example, transient expression of TALEN/CRISPR system could incorporate desired traits without stable integration of the transgene. Edited crops having non-detectable foreign DNA/RNA could face less opposition in regulatory/public clearance and could seem in line with their natural variants. NBTs could offer promising solutions to engineer complex genomic traits involving several molecular pathways like synthesis of starch, proteins, vitamins, etc., that otherwise would require tedious multistep engineering using conventional techniques. Other application may include functional studies of uncharacterized genes in potato using NBTs that could facilitate more precise and site-specific mutation at targeted loci. Furthermore, the induced mutations could be mapped by utilizing various next-generation sequencing (NGS) techniques. This could save the time for estimating off-site targeting effects that may or may not phenotypically appear later during crop growth. Conclusively, NBTs could be effectively used to engineer a number of nutritional traits in potato like enhanced protein content (Chakraborty et al., 2010), vitamin C content (Bulley et al., 2012), β-carotene level (Li et al., 2012), and others etc. ### GM POTATO COMMERCIALIZED SO FAR: RISK ASSESSMENTS AND REGULATIONS The expansion of biotech crops over last two decades has firmly established the role of genetic engineering in modulating various agronomical, environmental and predominantly health-related traits in plants. Today, more than two billion hectares of agricultural land is under cultivation of biotech crops, which signifies its importance and adaptability to meet future challenges through generating useful phenotypes in plants (Parisi et al., 2016). Most of the GM potato cultivars commercialized so far include trait incorporations such as resistance to viruses and other phytopathogens (Ricroch and Henard-Damave, 2016). The first GM potato appeared in the market in 1995 was named "NewLeaf " by Monsanto <sup>R</sup> , which was genetically engineered using a toxin Bt gene to generate resistance against Colorado beetle (Leptinotarsa decemlineata) (Kilman, 2001). Another engineered potato variety appeared in March 2010; a GM potato "Amflora," developed by BASF Plant Science and aimed at improved amylopectin content (waxy tuberous starch) for the processing industry, was approved by the European Commission (Lucht, 2015; Zaheer and Akhtar, 2016). A total of 23 GM potato lines are in the regulatory approval process (10 at precommercial, 11 at regulatory, and 2 at advance development stages) that has been engineered for various agronomical and quality related traits (Parisi et al., 2016). In 2017, the U.S. Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) has approved the cultivation of three GM potatoes (InnateTM Second-Generation; developed by Simplot corp. <sup>R</sup> ) meant to resist fungal (late blight) infections, and acrylamide formation (https://durangoherald. com/articles/140336-u-s-approves-3-types-of-geneticallyengineered-potatoes?wallit\_nosession=1). Importantly, these GM potatoes were developed using various modern breeding tools and got regulatory approvals. A number of other potato varieties engineered for various agronomic traits are in the pipeline or subjected to biosafety and field trials in different countries (Ricroch and Henard-Damave, 2016; Table 3). The development and commercialization of GM crops is a huge challenge for scientists and regulatory domains due to multiple technical, ethical and social/public limitations (Podevin et al., 2012). In addition, the socioeconomic benefit of utilizing these GM products is a big constraint to their producer and consumer adaptations. Still, the costs related to GM development/approval usually exceed \$35 million (Smyth et al., 2017), which seemed to be unsuitable for many low-income public-private institutions. This limits the interest of investing in GM technology in many developing countries (Pérez-Massot et al., 2013). The public acceptance in adopting GM-labeled products further makes it questionable for legislation, government and/or environmental authorities. With technological advancement, the NBTs could be used to generate a highly specific genetic modification that is indistinguishable FIGURE 1 \| A schematic diagram of new breeding technologies (NBTs) application for editing potato genome for nutritional improvement. (A) Clustered regularly interspaced short palindromic repeat/CRISPR associated9 (CRISPR/Cas9) system. Expression of constructs containing a single guide RNA (sgRNA) and Cas9 endonuclease will result in the assembly of sgRNAs and Cas9 nuclease to make a sgRNA/Cas9 complex. The designed sgRNA having sequence complementarity will bind specifically to a targeted site on genomic DNA and sgRNA/Cas9 complex will cleave 3' upstream of PAM (protospacer adjacent motif) sequence; shown by black scissors. This cleavage will result in double-stranded brakes (DSB) in targeted genome. (B) Transcription activator-like effector nucleases (TALENs) system. The TALE array contains a highly conserved (33–34 nt) DNA binding domain having repeat variable di-residues (RVDs) at positions 12 and 13 to guide the target-specific binding. Nuclease activity is performed by domains containing FokI endonucleases to produce DSBs. These DSBs are normally repaired by host-mediated DNA repair mechanisms which might results in targeted mutation and end in either gene disruption, correction or addition. The black circles having white text (1,2) represent the CRISPR/Cas9/TALENs cleavage of two host genes (vacuolar invertase, VInv; sterol side chain reductase, SSR2). (C) VInv is primarily involved in bioconversions of sucrose to fructose and glucose inside cell vacuole, precursors of acrylamide formation. (D) Biosynthesis of steroidal glycoalkaloids in plant cell from cycloartenol which is mediated by the activity of host SSR2 gene. NBTs-mediated targeting of host genes will result in reduced formation of anti-nutrients (acrylamide and steroidal glycoalkaloids) inside tubers and thus result in the improved quality of potato tubers. The proposed challenges (rectangles) by using these technologies might result in some questions such as society and regulation regimes' approval for editing food crop, off-site targeting effects on plants, the presence of any transgene, biosafety trails to check health-related issues, and the potential risks of horizontal gene transfer by using these GM crops. These questions need to be addressed while before using some NBTs. from natural variants/mutants and therefore significantly reduce the GM concerns (Wolt et al., 2016). For example, Cellectis <sup>R</sup> (a multinational biotech company) developed a transgenefree potato in 2014 engineered for improved processing traits (Wolt et al., 2016). They used TALEN technology to introduce a base deletion in potato genome through protoplast transformation of an exogenous genetic material from some plant pest (Phytophthora infestans). Phenotypic and molecular confirmation of regenerated products showed no detection of any exogenous material in segregating generation, thus, being transgene-free, APHIS (a USDA regulatory domain under the Plant Protection Act: CFR 7) did not take it under regulatory process (Wolt et al., 2016). ### CONCLUSION AND FUTURE PROSPECTS Research focusing on food safety and security can provide substantial ways to meet up the rising food demands, especially in the food-deficit countries. The rapid development of plant genetic engineering has provided new exciting tools to generate crops with enhanced yield and nutritional traits. In this context, potato crop has enormous potential to contribute to food security as it could provide low-cost, high energy food at sustainable basis (Zaheer and Akhtar, 2016). Several studies have demonstrated the incorporation of nutritional traits in potato such as enhanced protein content (Chakraborty et al., 2010), vitamin C content (Bulley et al., 2012), β-carotene level (Li et al., 2012), triacylglycerol (Hofvander et al., 2016), tuber methionine (Kumar and Jander, 2017), and amylose content (Krunic et al., 2018; Table 1). Other research priorities are given to reduce anti-nutritional compounds in potatoes such as steroidal glycoalkaloids (Itkin et al., 2013), acrylamide (Clasen et al., 2016) and other food toxins (Hajeb et al., 2014; Table 1). Recently, the emergence of NBTs such as TALENs, ZFNs, CRISPR/Cas9 etc. has provided opportunities for a robust, precise, and site-specific genome editing to introduce important agronomical traits in various crop plants (Mahfouz et al., 2016; Weeks, 2017). Within the context of potato genome editing, ongoing research is focused on utilizing NBTs to incorporate important traits (Table 2). However, most of these efforts generated end products having transgenic tags, being questioned by food safety, legislation, and extensive consumer opposition. To circumvent these regulatory barriers, NBTs research should now focus on generating transgene-free products, specifically in case of food crops (Wolt et al., 2016). Since, in vegetatively propagated crops like potato, the procedure for transgene removal in subsequent generations through segregation is time-consuming, the utilization of agroinfiltration and protoplast transformation to deliver NBTs' reagents provide a rational procedure for transgene-free potato production (Bortesi and Fischer, 2015). The CRISPR/Cas9 approach can be utilized to incorporate nutritional improvement in potato coupled with late blight resistance through transient expression of transcription factor (StWRKY1) in a transgene-free method (Yogendra et al., 2015). Other research priorities could focus on eliminating allergen compounds in potato such as alkaloids, glycoprotein patatin etc. (Zaheer and Akhtar, 2016). In addition, incorporation of abiotic (environmental, salinity, drought, temperature) stress resistance coupled with increased nutrition could facilitate potato to acclimatize in diverse agro-ecological zones, thus ### REFERENCES impeding food-shortage in less fertile/water deficit agricultural lands. The introduction of pest resistance into commercial cultivars would reduce the pesticide applications, thus impeding the environmental pollution. Further expansion of nutritional studies can set some preliminary values to justify the health benefits of potato-derived foods. Research efforts are needed to mitigate the mechanisms of nutrient-loss, such as copigmentation and to enhance the health-promoting components such as antioxidants and phytochemicals in commercial cultivars of potato. The availability of potato genome sequence (www. potatogenome.net) has facilitated the comparative genomic analyses to identify the genes useful for improving several agronomically important traits like tuberization, loss of bitterness, and diseases resistance (Hardigan et al., 2017; Li et al., 2018). The NBTs offer fast-track development of commercial potato cultivars such as Russet Burbank, Désirée, Kathadin etc. with superior traits such as improved nutrition, biotic and abiotic stress tolerance, and enhanced yield. However, to achieve such goals, it is paramount to acknowledge that not a single GE approach is sufficient to incorporate all the desired traits, rather an integration of NBTs coupled with well-established conventional breeding techniques will be needed. Here, we believe that the future of GM potato is reliant not only on some consumer-oriented traits such as fortified nutrition, enhanced flavor and appearance, but also on some industrial traits such as enhanced starch quality, and reduced CIS activity, which will ultimately enhance the marketability and long-term acceptability of GM potato. ### AUTHOR CONTRIBUTIONS SM provided the outlines of the review and contributed the key ideas. AH, SS and SZ wrote the manuscript and prepared the figures. SM, AH, and SZ worked on and improved the original draft and figures. The manuscript was approved by all co-authors. ### ACKNOWLEDGMENTS The authors would like to thank Dr. Ghulam Mustafa for valuable suggestions, and critically reviewing this manuscript. in the cultivated potato. Proc. Natl. Acad. Sci. U.S.A. 114, E9999–E10008. doi: 10.1073/pnas.1714380114 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Hameed, Zaidi, Shakir and Mansoor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach Grace Z. H. Tan<sup>1</sup> , Sudipta S. Das Bhowmik <sup>1</sup> , Thi M. L. Hoang<sup>1</sup> , Mohammad R. Karbaschi <sup>1</sup> , Hao Long<sup>1</sup> , Alam Cheng<sup>1</sup> , Julien P. Bonneau<sup>2</sup> , Jesse T. Beasley <sup>2</sup> , Alexander A. T. Johnson<sup>2</sup> , Brett Williams <sup>1</sup> and Sagadevan G. Mundree<sup>1</sup> \* <sup>1</sup> Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD, Australia, <sup>2</sup> School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia #### Edited by: Michael A. Grusak, Children's Nutrition Research, Agricultural Research Service, United States Department of Agriculture, United States #### Reviewed by: Marta R. M. Lima, University of California, Davis, United States Hamid Khazaei, University of Saskatchewan, Canada \Correspondence: Sagadevan G. Mundree [email protected]* #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 13 February 2018 Accepted: 24 May 2018 Published: 14 June 2018 #### Citation: Tan GZH, Das Bhowmik SS, Hoang TML, Karbaschi MR, Long H, Cheng A, Bonneau JP, Beasley JT, Johnson AAT, Williams B and Mundree SG (2018) Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach. Front. Plant Sci. 9:788. doi: 10.3389/fpls.2018.00788 Iron deficiency currently affects over two billion people worldwide despite significant advances in technology and society aimed at mitigating this global health problem. Biofortification of food staples with iron (Fe) represents a sustainable approach for alleviating human Fe deficiency in developing countries, however, biofortification efforts have focused extensively on cereal staples while pulses have been largely overlooked. In this study we describe a genetic engineering (GE) approach to biofortify the pulse crop, chickpea (Cicer arietinum L.), with Fe using a combination of the chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes which function in Fe transport and storage, respectively. This study consists of three main components: (1) the establishment for baseline Fe concentration of existing germplam, (2) the isolation and study of expression pattern of the novel CaNAS2 gene, and (3) the generation of GE chickpea overexpressing the CaNAS2 and GmFER genes. Seed of six commercial chickpea cultivars was collected from four different field locations in Australia and assessed for seed Fe concentration. The results revealed little difference between the cultivars assessed, and that chickpea seed Fe was negatively affected where soil Fe bioavailability is low. The desi cultivar HatTrick was then selected for further study. From it, the CaNAS2 gene was cloned and its expression in different tissues examined. The gene was found to be expressed in multiple vegetative tissues under Fe-sufficient conditions, suggesting that it may play a housekeeping role in systemic translocation of Fe. Two GE chickpea events were then generated and the overexpression of the CaNAS2 and GmFER transgenes confirmed. Analysis of nicotianamine (NA) and Fe levels in the GE seeds revealed that NA was nearly doubled compared to the null control while Fe concentration was not changed. Increased NA content in chickpea seed is likely to translate into increased Fe bioavailability and may thus overcome the effect of the bioavailability inhibitors found in pulses; however, further study is required to confirm this. This is the first known example of GE Fe biofortified chickpea; information gleaned from this study can feed into future pulse biofortification work to help alleviate global Fe deficiency. Keywords: pulse biofortification, iron, genetic modification, nicotianamine synthase, soybean ferritin, crop improvement, chickpea (Cicer arietinum L.) ### INTRODUCTION Iron (Fe) deficiency has long been recognized as one of the most common micronutrient deficiencies in the world. Afflicting both developing and developed nations, it is the cause of more than 60% of global anemia cases (WHO, 2008; Alvarez-Uria et al., 2014). To combat this problem several strategies have been developed such as dietary diversification, supplementation, food fortification, and crop development. Amongst these, the development of crops with increased Fe concentrations and/or bioavailability (also known as "biofortification") has garnered great interest due to its sustainability, cost-effectiveness, and accessibility of products to vulnerable populations (Nestel et al., 2006). Biofortification can be achieved through breeding or genetic engineering (GE), and this has been performed in various crop species. The focus thus far, however, has mostly been on starchy staples like as cereals (e.g., rice, wheat, pearl millet) and root crops (e.g., potato, cassava) (HarvestPlus, 2015). Naturally, it is in these species that the greatest advances have been made. For instance, more than three-fold increase in Fe concentration has been reported in biofortified pearl millet and its effectiveness in combating Fe deficiency anemia has been verified via feeding trials (Cercamondi et al., 2013; Finkelstein et al., 2015). Aside from the aforementioned staples, recent years have seen growing interest in pulses as targets for Fe biofortification. Pulses are defined as leguminous crops harvested solely for dry grain (FAO, 1994), and most serve as important secondary staples, particularly with their high protein content (Iqbal et al., 2006); it is this latter feature that also complements the existing biofortification work in cereals. The pulse biofortification effort is relatively young compared to cereals but there has been considerable progress, notably in the common bean. Several biofortified varieties have been generated from the HarvestPlus breeding programs, with up to 94% enhancement in seed Fe concentration achieved (Katsvairo, 2015). The work has also progressed to feeding trials which have yielded promising results (Cercamondi et al., 2013; Kodkany et al., 2013; Finkelstein et al., 2015). This success has paved the way for advances in other pulses. Studies on Fe accumulation traits have been performed on cowpea (Fernandes Santos and Boiteux, 2015), chickpea (Diapari et al., 2014), pea and lentil (Ray et al., 2014), and test trials are currently underway for some of them (HarvestPlus, 2016). Thus far, most, if not all, of this work has been focused on breeding while GE remains unexplored. As such, there are no established GE strategies for pulses, though some lessons can be drawn from the work in cereals. One of the most successful examples to date is the GE Fe biofortified rice (Orzya sativa), in which seed Fe concentration was increased by 7.5-fold with no yield penalty (Trijatmiko et al., 2016). The strategy targeted the three core processes of Fe metabolism—uptake, translocation, and storage—through constitutive overexpression of the rice Nicotianamine Synthase 2 gene (OsNAS2) gene and seed-specific expression of the soybean (Glycine max) Ferritin (GmFER) gene. NAS catalyzes the biosynthesis of nicotianamine (NA), a nonproteogenic chelator of divalent transition metals that facilitates translocation of said metals in plants (Scholz et al., 1992). In graminaceous species, it is also a precursor for the mugeneic acid (MAs) family of phytosiderophores which contribute to both Fe uptake from soil and in planta translocation (Higuchi et al., 1999). When constitutively overexpressed in rice cv. Nipponbare, the OsNAS2 gene caused a four-fold increase in grain Fe concentration (Johnson et al., 2011). Ferritin (FER), on the other hand, is an Fe storage protein that allows for safe sequestration of Fe in a soluble and bioavailable form. When overexpressed in the seed, GmFER has been demonstrated to increase seed Fe concentration by up to threefold in several plant species (Goto et al., 1999). Excessive expression, however, may lead to disproportionate sink strength, resulting in altered sequestration of Fe in source tissues and the development of Fe deficiency symptoms (Van Wuytswinkel et al., 1999; Qu et al., 2005; Masuda et al., 2013b). This problem can be rectified by increasing Fe uptake and translocation capacities, such as through the co-expression with NAS (Masuda et al., 2013a). In this case, a synergistic effect was also achieved, producing greater enhancement in seed Fe concentration (Wirth et al., 2009; Trijatmiko et al., 2016). Whether this strategy will have a similar effect on Fe concentration when applied to a pulse crop is uncertain. However a major advantage is its potential effect on Fe bioavailability. Both NAS and GmFER have been linked to increased Fe biovailability (Davila-Hicks et al., 2004; Lönnerdal et al., 2006; Zheng et al., 2010), a feature not usually accorded to other commonly used Fe metabolism genes. This is particularly relevant to pulse biofortification given the inherently high levels of antinutrients like phytic acid which inhibit Fe absorption in the gut (Sandberg et al., 1989; Hemalatha et al., 2007; Petry et al., 2014). For this study, target species is chickpea (Cicer arietinum). The second most important pulse crop in the world with an annual production exceeding 14.2 million tons (FAO, 2016). The bulk of the chickpea crop is currently grown and consumed in India where human Fe deficiency is prevalent, however, continued population growth is likely to result in increased demand for chickpea in Africa and other parts of Asia (Rao et al., 2010; Akibode and Maredia, 2012). Fe concentrations in chickpea has been found to range from 3 to 14.3 ppm (Wood and Grusak, 2007), though due to the presence of naturally occurring inhibitors, only a small fraction is bioavailable (Hemalatha et al., 2007). Both iron concentration and bioavailability is subject to genotype and environmental effects, and to date, detailed studies of such effects are limited to populations in India (Upadhyaya et al., 2016) and Canada (Diapari et al., 2014). No such information is available for Australian populations, and part of this study would therefore serve to partially fill in this gap. Overall, the main aim of this study was to biofortify chickpea by GE to overexpress NAS and GmFER. This body of work consisted of three parts: (1) assessing the macroand micro-elemental composition of six modern Australian chickpea cultivars and identifying a suitable cultivar for Fe biofortification research, (2) cloning and expression analysis of an endogenous chickpea NAS gene termed CaNAS2, and (3) constitutive overexpression of the CaNAS2 gene and constitutive expression of soybean GmFER in chickpea as a novel GE approach to produce Fe biofortified chickpea. ### MATERIALS AND METHODS ### Plant Material For the elemental composition analysis of commercial chickpea, three kabuli cultivars (Genesis090TM, KalkeeTM, and PBA Monarch) and three desi cultivars (PBA Boundary, CICA0912, and PBA HatTrick) were used. Seed samples were obtained from field trials at four locations within Queensland—Billa Billa, Warra, Roma, and Kingaroy—and from the seed company Grainland in Moree, New South Wales. All seeds were produced during the 2014 winter growing season. Information on the cultivation sites and conditions during the growing period, where available, is listed in Supplementary Table 1. The soil types of the field locations were provided by Dr Yash Chauhan from the Agricultural Production Systems sIMulator (APSIM) database. For the gene expression analyses and chickpea transformation, PBA HatTrick seeds were purchased from the seed company Grainland in Moree, New South Wales. ### Plant Growth Conditions All in vitro cultivation was performed in growth cabinets set at 24 ± 1 ◦C, under fluorescent lights with a 16 h light/ 8 h dark cycle. For glasshouse cultivation, temperatures were maintained at 21 ± 1 ◦C and 61 ± 1.5% relative humidity. Natural lighting was used except during dusk, when artificial lighting was then turned on to complete a 16 h light/ 8 h dark cycle. An average natural light intensity of around 450 ± 1 µmoles s-1 m-2 of cloudy and sunny day prevailed during the growth period. Seeds were first germinated in Plugger's potting mix, before transplanting to 400 × 250 mm pots containing a 1:1 mixture of University of California (UC) mix and Searles <sup>R</sup> Premium potting mix. The recipe for the UC mix consists of 80 kg sand, 120 kg peat, and 100 kg sand, peat, and gravel, supplemented with 400 g blood and bone, 100 g Micromax micronutrients, 40 g KSO4, 40 g KNO3, 400 g superphosphate, 300 g hydrated lime, and 1,200 g dolomite. Plant were watered with 100 mL every 2 days via an automated watering system. After ∼3 months, when at least 80% of the pods have filled, watering was ceased in preparation for harvesting. Harvesting was done approximately 3 weeks thereafter, or when the plants have completely dried. All seeds were de-husked by hand and stored in paper envelopes at 4◦C until planting or analysis. ### Elemental Analysis All samples were cleaned, freeze-dried, and milled prior to analysis. A minimum of three biological replicates were used per transgenic event. For leaf tissue, milled samples were pressed into 5 mm diameter pellets and analyzed via LA-ICP-MS (laser ablation inductively-coupled mass spectroscopy) using an Agilent 8,800 Inductively Coupled Plasma Mass Spectrometer attached with an ESI 193 nm Excimer Laser. The laser was set at a pulse width of 4 ns, spot size of 85 microns, and scan speed of 10 microns/s. At least three lines scans were used for each sample as technical replicates. For whole seed analysis, acid digestion was performed on milled samples. Briefly, 2 mL HNO<sup>3</sup> and 0.5 mL H2O<sup>2</sup> were added to 200–300 mg of milled sample, vortexed, and allowed to stand overnight at room temperature. Following digestion, the tubes were shaken at 200 rpm for 20 min, incubated at 80◦C for 30 min, then 125◦C for 2 h. Upon cooling to room temperature, the volume was made to 25 mL using MilliQ water and the samples agitated at 300 rpm for 5 min. Undissolved material (e.g., silicates) was settled for 60 min. The settled extract was then filtered and analyzed via ICP-OES (inductively coupled plasma optical emission spectroscopy) using a Perkin Elmer Optima 8300 DV Inductively Coupled Plasma Optical Emission Spectrometer. Three technical replicates were prepared per sample. For analysis of trace element distribution in the seed, 100 seeds were imbibed in MilliQ water for 20 h. The seeds were then separated into the seed coat, cotyledons, and radicle. To measure approximate distribution of mass, the weight of the individual parts of 10 seeds were taken. Tissues of the same type were then pooled and processed for analysis like the whole seed. Three technical replicates were prepared per sample. ### Designation and Bioinformatics Analysis of Chickpea NAS2 Gene Four chickpea NAS amino acid sequences (XP\_004495658.1, XP\_004487761.1, XP\_004488704.1 and XP\_004494544.1) were retrieved from the NCBI database. The ortholog with highest similarity to the amino acid sequence encoded by the rice OsNAS2 gene (LOC\_Os03g19420) was designated as the CaNAS2 gene coding for protein XP\_004495658.1 (Supplementary Table 2). The three additional NAS genes were named as the following: CaNAS3 coding for XP\_004487761.1 protein, CaNAS4 coding for XP\_004494544.1 protein, and CaNAS1 for XP\_004488704.1 protein respectively. The four NAS amino acid sequences were aligned based on amino acid conservation using the Geneious Pro 5.6.6, as per the settings described by Bonneau et al. (2016) (CLUSTALW—cost matrix BLOSUM62, TABLE 1 \| Summary of Fe, Zn, and P concentrations in kabuli and desi cultivars grown at different locations. All values are expressed as ppm. Data are expressed as mg/100 g and presented as a mean of all cultivars collected from that site. For each cultivar per site, n = > 3. Values with different superscript letters indicate a significant difference at p < 0.05. threshold 1). A blastn using the genomic sequences of the four CaNAS genes was performed against Cicer arietinum (cv. kabuli, CDC Frontier)—CDS database (https://legumeinfo. org) to identify chromosomal location. Several bioinformatics tools were then used to predict characteristics of the enzyme encoded by CaNAS2: the theoretical isoelectric point (pI) and molecular weight were calculated using the Compute pI/Mw tool on ExPASY (Bjellqvist et al., 1993, 1994; Gasteiger et al., 2005); the hydrophobicity profile of the protein was assessed using ProtScale (Gasteiger et al., 2005) and potential transmembrane sections were identified using TMpred (http://www.ch.embnet. org/software/TMPRED\_form.html). A check for motif sequences was conducted using ScanProsite (de Castro et al., 2006) and MOTIF Search GenomeNet<sup>1</sup> Phobius (Käll et al., 2004) and iPSORT (Bannai et al., 2001, 2002) was used to identify potential signaling peptides. ### Phylogenetic Analysis of NAS Proteins A progressive pairwise alignment was performed using full length amino acid sequences of 58 NAS proteins (Supplementary Table 3) using default settings of Geneious alignment (global alignment with free end gaps, Blosum62, gap open penalty 12, gap extension penalty 3)—Geneious Pro 8.1.7 software. Once the protein alignment, a phylogenetic analysis which generated an unrooted tree was conducted as described in Beasley et al. (2017). ### RNA Extractions and Quantitative RT-PCR Total RNA was isolated from frozen tissues using RNeasy Mini kits (QIAGEN) following the manufacturer's instructions. A 500 ng aliquot of total RNA was treated with RQ1-DNAse (Promega) and the absence of contaminating DNA confirmed via PCR. cDNA was then synthesized using SuperScriptTM IV Reverse Transcriptase (ThermoFisher Scientific). The synthesized cDNA was used for qualitative RT-PCR and quantitative RT-PCR. For qualitative RT-PCR, each reaction comprised of 5 µL of 2X GoTaq green (Promega), 0.25 µL each of 10µM forward and reverse primers, 0.6 µL of DMSO, and 1 µL of undiluted cDNA as the template. MilliQ water was added to reach a final reaction volume of 10.6 µL. The PCR program used was as such: initial denaturation at 95◦C for 3 min, followed by 30 cycles of denaturation at 95◦C for 30 s, annealing at 48–60◦C (depending on primers) for 30 s, and extension at 72◦C. Extension time was set at 1 min per 1 kbp of the final product size. A final extension was done at 72◦C for 5 min. For qPCR, a 1:30 dilution of cDNA was used as template. The latter was performed on a CFX384 TouchTM Real-Time PCR Detection System (BIO-RAD) using the SYBR Green PCR Master Mix kit (Applied Biosystems). A primer concentration of 300 nM was used, and the primer sequences are as listed in Supplementary Table 4. The housekeeping genesEF1α, and GAPDH were included in each run to serve as internal controls; their primer sequences are as published by Garg et al. (2010). All housekeeping genes were confirmed to be stable under the experimental conditions used. Three biological replicates were used, from each of which three technical replicates were <sup>1</sup>Available online at http://www.genome.jp/). Data is presented as a mean ± SD. All cultivars from had n=3 except for HatTrick from NSW, which has n = 5. Values sharing the same superscript letters indicate groups that are not significantly different at p < 0.05 when tested with one-way ANOVA, using Tukey's HSD post-hoc test. prepared. The qPCR program used was as follows: initial denaturation at 95◦C for 10 min, followed by 45 cycles of denaturation at 95◦C for 10 s, annealing at 60◦C for 30 s, and slow ramping of 0.5◦C/min from 65 to 90◦C for the melt curve. ### Fe Deficiency Experiment PBA Hattrick seeds were sterilized and germinated on half strength Murashige and Skooge (MS) media. Seed coats were removed post-germination and the seedlings were acclimatized for 4 days in tap water. Twenty four-week old seedlings of approximately the same size and developmental stage were transferred to a hydroponics system in a growth cabinet, with 10 replicates per set-up. Later, 1 month old plants were treated with full-strength Hoagland solution with or without Fe-EDTA. Each setup contained ∼600 mL of Hoagland solution which was topped up every 3 days. The Hoagland solution was replaced with MilliQ water every third top-up to dilute any accumulated salts. Visible chlorosis in the Fe-deprived plants was observed after 4 weeks of treatment and samples were collected 2 weeks thereafter. Three plants of similar conditions and growth stage were selected from each treatment and the following tissue types collected: mature leaf, stem, cotyledon, and root. Senescent leaf and chlorotic leaf were also collected from the Fe-sufficient and Fe-deficient plants respectively. All samples were snap-frozen in liquid nitrogen immediately after collection and stored in −80◦C until RNA extraction. ### Cloning of CaNAS2 and Construction of NAS-GmFER Overexpression Vectors A binary vector using the pOPT-EBX backbone was constructed to constitutively overexpress the CaNAS2 gene and constitutively express the GmFER gene (Figure 1). Included in the T-DNA region was the selectable marker gene neomycin phosphotranferase II (NPTII) which confers resistance to the antibiotics geneticin and kanamycin. All cloning primers used are listed in Supplementary Table 5. The CaNAS2 gene was cloned from chickpea (cv HatTrick) genomic DNA, with primers designed from the predicted sequence in the Genbank database, accession number XM\_004495601. Restriction sites were added to the ends via site-directed mutagenesis using high fidelity PCR (Phusion <sup>R</sup> , NEB). The amplified fragment was cloned into a pGEM <sup>R</sup> -T Easy, then subcloned TABLE 3 \| Pearson's correlation coefficient between the different trace elements in PBA HatTrick. For Billa Billa, Roma, Warra, and Kingaroy, samples were n = 3 while NSW was n = 5. Grain samples from four locations within Queensland (Billa Billa, Warra, Roma, and Kingaroy) and a seed supplier from New South Wales were analyzed by ICP-OES. Values marked with an \*Indicate a significant correlation between two elements (p < 0.05). in a 5′ to 3′ direction to a pGEM <sup>R</sup> -T Easy vector with a cassette containing a Nos promoter and CaMV 3′ UTR. Following this the NosP-CaNAS-CaMV 3′ UTR was digested and ligated to a pOpt-EBX-GmFER backbone to form the complete vector. To generate the pOpt-EBX-GmFER backbone, GmFER was cloned from a synthesized fragment (accession no. NM\_001250105.2). The amplified fragment was cloned into a pGEM <sup>R</sup> -T Easy vector, then subcloned into the pOpt-EBX backbone containing a CaMV 35s promoter and Nos terminator. To ensure integrity and correct orientation of each gene and component, sequence verification was performed after each cloning step in the above process. The final verification was performed on the completed construct, which was then transformed into electrocompetent Agrobacterium tumefaciens strains Agl1. ### Generation and Molecular Characterisation of Transgenic Chickpea Agrobacterium-mediated transformation of the desi cultivar, PBA Hattrick, followed the protocol developed by Sarmah et al. (2004) with a few modifications. Briefly, half-embryonic axes were prepared from imbibed seeds. Additional injury was inflicted to the cut surface of the radicle using a sterile 26 gauge needle dipped in Agrobacterium strain AGL-1 harboring the expression vectors. The explants were immersed in Agrobacterium for an hour, followed by co-cultivation in B5 media for 72 h. Following co-cultivation, the explants were transferred to regeneration and selection medium 1 (MS media containing 500 µg/L of BAP, 500 µg/L of kinetin, 50 µg/L of NAA, 200 mg/L of kanamycin and 25 mg/L of meropenem). Shoots obtained in first round of regeneration and selection medium were further selected by subsequent subculturing in the regeneration and selection medium 2 (MS media containing 500 µg/L of BAP, 500 µg/L of kinetin, 200 mg/L of kanamycin and 25 mg/L of meropenem) every 14–21 days. Up to eight rounds of selection were done to obtain putative GE events. Any explants that exhibited proliferative shoot growth during that duration were isolated and considered an individual GE event. Upon reaching an appropriate size, shoots from such multiplying clumps were grafted onto non-GE rootstocks grown on half-strength MS media. Grafts were allowed to set for up to 3 weeks before acclimatization. Acclimatized plants were screened via PCR of gDNA for the genes of interest. To avoid false negatives caused by potential chimerism, a pooled sample consisting of leaves from every branch was used. Primers used for PCR screening are as listed in Supplementary Table 6. Two PCR-positive T<sup>0</sup> events were propagated to maturity for two generations to obtain sufficient T<sup>3</sup> seed for the experiments described in this paper. Null segregants from every generation were also maintained to serve as negative controls. ### NA Quantification Freeze-dried seeds from three different plants of the same transgenic event were pooled and milled to form a bulked flour sample, from which four technical replicates were drawn for NA quantification. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify 9-fluorenylmethoxycarboxyl chloride (FMOC-Cl) derivatized NA on an LC 1290 series coupled to a 6490 series triple quadrupole MS (Agilent Technologies Inc.) using established protocols (Selby-Pham et al., 2017). In short, a combined methanol (100%) and 18 M H2O extraction (5 µL) of pulverized chickpea flour (25 mg) was combined with sodium borate buffer (pH = 8, 1 M, 10 µL), EDTA buffer (pH = 8, 50 mM, 10 µL), and fresh FMOC-Cl solution (50 mM, 40 µL). After incubation (60◦C, 700 rpm, 15 mins), the solution mixture was quenched via the addition of formic acid (pH = 4, 5%, 8.9 µL). Chromatography was performed using a reversephase column (Zorbax Eclipse XDB-C18, HS 2.1 × 100 mm 1.8 Micron, Agilent Technologies Inc.) with aqueous (0.1% v/v FA in dH2O) and organic (0.1% v/v FA in acetonitrile) mobile phases. ### Statistical Analysis All statistical analysis was performed on Minitab statistical software (Arend, 2010) using one-way ANOVA. The Tukey HSD Data are presented as a mean ± SD. All cultivars from had n = 3 except for HatTrick from NSW, which has n = 5. Values sharing the same superscript letters indicate groups that are not significantly different at p < 0.05 when tested with one-way ANOVA, using Tukey's HSD post-hoc test. FIGURE 2 \| Distribution of macro and micro elements in chickpea (cv HatTrick) seed expressed as a percentage of the total element content in the three main tissue types present in the grain. Values for the elemental profile was derived from a bulked flour sample produced from pooling tissue from 100 seeds. Three technical replicates were used. Mass distribution in the seed was calculated from 10 biological replicates. represent the YXXΦ and the LL, IL, or ML motifs. this tree are Arabidopsis thaliana (AtNAS), Hordeum vulgare (HvNAS), Lotus japonicus (LjNAS), Medicago truncatula (MtNAS), Oryza sativa (OsNAS), Thlaspi caerulescens (TcNAS), Solanum lycopersicum (SlNAS), Zea mays (ZmNAS), and Triticum aestivum (TaNAS). Black nodes (•) represent weak bootstrap values (<75%). The scale bar corresponds to branch length and longer branches correspond to greater numbers of nucleic acid polymorphisms along the sequence. test was used in the analysis the different chickpea cultivars, while Dunnett's test was used in the analysis of the GE chickpea. ### RESULTS ### Mineral Composition of Chickpea Cultivars and Identification of Factors Influencing Seed Fe Concentration Fe concentrations in chickpea were found to range from 3.36 to 52.0 ppm, with no significant differences between the cultivars, though average values were slightly higher in the kabuli types compared to the desi (Tables 1, 2). The highest values were noted in the kabuli cultivar Genesis090TM, while the lowest values were mostly found in PBA HatTrick, though the difference to other desi cultivars was negligible (Table 2). This, in combination with the availability of established transformation protocols for the cultivar, made HatTrick the choice candidate for further work. Between locations, similar mineral profiles were observed amongst samples grown in Billa Billa, Roma, and Warra, which had vertosol-type soils. In contrast, samples obtained from Kingaroy contained less Fe. Also, unique to this locality was a high Mn concentrationto Fe ratio, which appears to have produced the negative correlation between the two elements (Table 3). No other negative correlations were observed between Fe and other elements. On the other hand, the strongest positive correlations were found between Fe, Zn, and P. Zn and P concentrations in particular. Unlike Fe however, greater differences in Zn and P were observed between the locations than between the genotypes (Table 4). ### Cotyledons Serve as the Primary Store for Fe in PBA Hattrick Seeds Amongst the different part of the seed, the radicle was found to have the highest Fe concentration at 95.0 ppm, followed by the cotyledons at 50.0 ppm. Due to its small mass however, its contribution only 3% to the total seed Fe content. The cotyledons on the other hand, constituted the bulk of the seed mass and contained 90% of the seed's total Fe. It was also the main store for all the other elements tested. The exception to this was calcium and manganese—the bulk of the former was found in the seed coat, while the latter was almost equally divided between the seed coat and cotyledons (Figure 2). ### Legume NAS Homologs Form Distinct Branches Among the Non-graminaceous Orthologues Concerning their chromosomal locations, the CaNAS2 and CaNAS4 genes were located on chromosomes 4 (Ca4) and 3 (Ca3) respectively, while both CaNAS1 and 3 were located on chromosome 1 (Ca1). All four CaNAS genes were found to be to consist of a single exon. CaNAS1, 2, 3, and 4 coded for 285, 306, 311, and 318 amino acids respectively. In the CaNAS1 protein, a longer N-terminal and a shorter C-terminal was seen compared to the other three CaNAS homologs. In the four CaNAS amino acid sequences, several highly conserved regions were noted (Figure 3). Of these, the YXX8 (Y refers to tyrosine, X to any amino acid residue, and 8 to bulky hydrophobic residues) and di-leucine (LL; leucine may be substituted with isoleucine) motifs were known to be conserved amongst the NAS homologs. Amongst some of the legume NAS sequences however, a variation of the LL motif was observed where the first leucine was substituted by methionine. This was seen in the CaNAS3 protein sequence, as well as in MtNAS2 from Medicago truncatula and LjNAS2 from Lotus japonicus (Supplementary Table 3). Phylogenetic analysis of the 58 NAS proteins revealed a clear distinction between graminaceous and non-graminaceous sequences (Figure 4). Two clades were present in the former and were consistent with a prior report (Bonneau et al., 2016). With the latter, three subgroups for legumes were observed these were defined as subgroups 1, 2, and 3. Subgroup 1 consisted of includes CaNAS1 from chickpea and MtNAS1 from Medicago truncatula. Subgroup 2 consisted of CaNAS3, MtNAS2, and LjNAS2 (from Lotus japonicus). Lastly, Subgroup 3 consisted of CaNAS2 and 4, MtNAS3 and 4, and LjNAS1. The presence of CaNAS2/MtNAS3 and CaNAS4/MtNAS4 in the same branch is most likely due to genome duplication. Further bioinformatic analysis of the CaNAS2 protein indicated an approximate molecular weight and pI of 34.36kDA 5.52 respectively. The enzyme was mostly hydrophilic with a potential transmembrane domain at position 126–151. A noncytoplasmic localisation was predicted, though no apparent signaling peptides were detected. ### CaNAS2 Is Expressed in Various Vegetative Tissues Under Fe Sufficient Conditions The expression pattern of CaNAS2 was examined under Fesufficient and Fe-deficient conditions. The Fe-deficient plants used in this study were observed to be paler green than the Fe sufficient controls, with severe chlorosis in the young leaves. No nodules were observed in either Fe-deficient or Fesufficient plants. CaNAS2 transcripts were detected in all the tissue types tested, though the levels were largely influenced by Fe status, with an overall downregulation under Fe deficiency. Gene expression in the Fe deficient plants, where detected, was generally low, and comparable across all tissues (Figure 5). Similar levels were also detected in the senescing leaf of Fesufficient plants. By contrast, other Fe sufficient tissues exhibited markedly higher expression, particularly in the stem, cotyledons, and roots. A 16-fold difference was seen between stems of the Fe-sufficient and Fe deficient plants, while expression was only detected in Fe-sufficient cotyledons. For root tissue however, no data could be obtained for the Fe deficient plants due to the consistently poor quality of the extracted RNA. ### Transgenic Chickpea Highly Express Both Transgenes and Have Increased Fe in Leaf Tissue and Increased Nicotianamine in Seed Tissue Both of the regenerated GE events (6.1 and 6.14) that were propagated to the T<sup>3</sup> generation were confirmed via Southern blot to have single transgene integration sites. Expression analyses showed a 49- and 93-fold increase in CaNAS2 expression in events 6.6 and 6.14, respectively, compared to null segregant controls (Figure 6B). Expression analysis showed an 18- and 30 fold increase in expression of GmFER in events 6.6 and 6.1, respectively, compared to null segregant controls (Figure 6A). Agronomic performance of the T<sup>3</sup> GE events in the glasshouse was generally comparable to the null controls and no significant differences were seen in terms of morphology and the other parameters measured (Figures 7, 8). Two differences were observed with respect to micronutrient composition of the leaf tissue of events 6.6 and 6.14 compared to null segregant controls (Figure 9). Event 6.6 had significantly higher leaf Fe concentration which was 1.39-fold higher than the null segregant control. Event 6.14 had significantly lower leaf Zn concentration which was 1.4-fold lower than the null segregant control. No significant differences for Fe, Zn, or Mn concentrations were observed in the seed of events 6.6 and 6.14. Seed from events 6.6 and 6.14 contained significantly higher flowering/pod-filling stage. NA concentration which was nearly 2-fold higher than the null segregant control (Figure 10). ## DISCUSSION ### Conditions Affecting Seed Fe Concentrations in Chickpea and the Selection of cv PBA Hattrick for Fe Biofortification Research Australian-grown chickpeas were previously reported to contain up to 140 ppm of Fe in their seed, though average values were ∼50 ppm (Petterson and Mackintosh, 1994). Such average values appear to be the norm globally, with similar results reported in chickpea from other countries (Meiners et al., 1976; Jambunathan and Singh, 1981; Thavarajah and Thavarajah, 2012; Diapari et al., 2014; Ray et al., 2014). That similar average values for seed Fe concentrations were obtained for the six cultivars used in our study, indicating that the cultivars used fell within that global norm. using Dunnett's post-hoc test (p 0.05). A past study by Ray et al. (2014) has reported seed Fe concentration to be influenced firstly by environmental conditions, then by genotype. Our observations were only partially consistent with that report, perhaps due to the smaller number of locations and cultivars we examined. Between the cultivars and most locations, no major differences were noted. The major environmental effect on seed Fe was only observed where soil Fe bioavailability was potentially compromised, such as with the Kingaroy samples which had lower seed Fe concentrations. Past records have shown Kingaroy ferrosols to be acidic with a high manganese to Fe ratio, and such conditions have been documented to inhibit Fe uptake, sometimes to the point of chlorosis (Twyman, 1951; Tanaka and Navasero, 1966; Alvarez-Tinaut et al., 1980). In our study, this inhibition was asymptomatic; no Fe deficiency symptoms were reported by the growers, and the effect was only apparent upon assessment of the seed mineral profile. This is potentially problematic where biofortification efforts are concerned as attempts to increase Fe concentrations may be unknowingly hijacked by adverse soil conditions. An environmental effect was also apparent in the Zn and P concentrations, both of which were positively correlated with Fe and affect seed nutritional value. With Zn, significant variations were noted between all sites regardless of cultivar, corresponding with observations by other authors who also reported significant year to year variations, even with seed from the same sites (Diapari et al., 2014; Ray et al., 2014). Management practices may explain at least part of the higher degree of Zn variation compared to Fe. Application of Zn fertilizers is a recommended practice in chickpea cultivation due to the risk of Zn deficiency in most Australian soils (Norton, 2013; Pulse Australia, 2016). The effects of Zn fertilization on grain Zn concentration however, are unpredictable and may differ between seasons (Akay, 2011). This variability is compounded by the effect of other management regimes like the application of P fertilizer. Aside from directly affecting grain P concentration (Saastamoinen, 1987), studies in pearl millet and wheat have highlighted a negative impact of P fertilizer on seed Zn concentration (Buerkert et al., 1998; Ryan et al., 2008). The precise reason behind it is uncertain, though it has been attributed to altered zinc uptake and the dilution effect caused by increased yields. The former was deemed the more likely, given the absence of adverse effects on seed Fe concentration (Ryan et al., 2008), though regardless, the implications on the nutritional quality of the seeds are still considerable. Seed P is primarily stored as phytate (Lolas et al., 1976; Griffiths and Thomas, 1981; Ravindran et al., 1994), a potent inhibitor of Fe and zinc bioavailability (Turnlund et al., 1984; Sandberg et al., 1989, 1999), and its levels can be considered a crude indicator of micronutrient bioavailability. In terms of the genetic effect, only a slight influence was seen. Few significant differences were found amongst the cultivars assessed, though kabuli cultivars had marginally higher Fe concentrations than the desi. This lack of difference is likely a product of the breeding process. Currently, no information exists for micronutrient accumulating traits in existing germplasm. As micronutrient accumulation is often accompanied by yield penalties (Garvin et al., 2006; Ficco et al., 2009; Diapari et al., 2014), it is likely that such traits may have been bred out of the current cultivars as breeding efforts in Australia have primarily focused on yield, abiotic stress, and biotic stress resistance with no consideration for nutritional value (Pulse Breeding Australia, 2017). Consequently, reintroduction of Fe-accumulation traits may prove challenging, though the difficulty can be alleviated with modern biotechnology. For this purpose the cultivar with the lowest Fe concentrations, PBA HatTrick, was identified as a suitable candidate for Fe biofortification. The benefits of this choice are manifold. PBA HatTrick is a popular choice amongst growers due to its high yield and resistance to phytophthora root rot. As a desi cultivar, it also has great potential for widespread dissemination, as desi constitutes 90% of the Australian chickpea export and therefore the bulk of the international market (Pulse Australia, 2016). With Fe localized primarily to the cotyledon, which is the main product, enhancements in Fe concentration will reach the consumer regardless of the form in which the seed is consumed. Bioavailability, however, may be a concern as phosphorus (and by extension, phytate) co-localizes with Fe to the cotyledons. This may perhaps be addressed with appropriate biofortification strategies and choice of target genes, one of which, for the purposes of this study, has identified as the novel CaNAS2 gene. ### CaNAS2 Has a Potential Housekeeping Role Under Fe Sufficient Conditions In our study, CaNAS2 grouped with the other dicot sequences in a separate clade to monocot sequences. This dichotomy between the dicot and monocot sequences is consistent with the findings of other authors (Hakoyama et al., 2009; Filipe de Carvalho et al., 2012), and is likely reflective of the differing physiological roles of NAS between the two. However whether this is conclusive remains to be seen due to the limited number of experimentally verified homologs; monocot sequences used in this study were of graminaceous origins, and the inclusion of the non-graminaceous homologs may potentially alter the existing arrangment. Nonetheless recent evidence have revealed functional grouping amongst NAS homologs (Bonneau et al., 2016) in terms of roles in development and Fe deficiency. Assuming this is universal amongst higher plants, such grouping will allow for accurate prediction of the function of closely related homologs. This accuracy however, remains subject to the availability of sequences that can be extrapolated from–such is evident in this study. For example, nodule-specific expression of LjNAS2 (Hakoyama et al., 2009) was neither mirrored in the closely related MtNAS2 (Medicago truncatula Gene Expression Atlas, 2014), nor in the any of the other legume NAS used in this study. Whether the third member of Subgroup 2, CaNAS3, will be nodule-specific is uncertain—this could not be investigated in our study due to the absence of nodules as the plants were not inoculated with Rhizobium. Still, given that the substitution of the di-leucine motif by a methionine is a trait unique to Subgroup 2, it is plausible that some functional specialization is present. Further investigation and discovery of more nodulespecific homologs may shed more light on this. Functional specialization may also be present in Subgroup 1, to which CaNAS1 and MtNAS1 belong. The nature of this specialization is still inconclusive. Subgroup 1 was the most divergent from other NAS proteins used in the phylogenetic analysis and no discernible trend could be seen between members. No orthologue of CaNAS1 was found in Lotus japonicus, perhaps due to the genome evolution in legumes (Wang et al., 2017). As with Subgroup 2, further study is required before conclusive statements may be made. In the interim, only Subgroup 3 bears enough information for reasonable inference of function. Subgroup 3 homologs are notable for their widespread expression in various vegetative tissues (Hakoyama et al., 2009; Medicago truncatula Gene Expression Atlas, 2014), and such is also seen in CaNAS2 and 4 (Figure 5, Supplementary Figure 1). While the expression sites vary between homologs, ranging from roots, to leaves, and cotyledons, a common feature is the expression in the stem. Using LjNAS1 as a reference, this pattern may indicate a housekeeping role in the systemic redistribution of Fe (Hakoyama et al., 2009) which, in the case of CaNAS2, occurs under Fe-sufficient conditions. It is also likely that the expression of NAS in the diverse sites may operate at different scales, given the involvement of NA in long and short-distance translocation. Expression in the stem may serve to feed NA into the vascular tissue and symplast for systemic transport, while expression in the other locations may provide NA for more localized translocation. Concerning the movement and localization of CaNAS2 within an intracellular context, the results predicted a noncytoplasmic, and potentially vesicular, localization. The accuracy of this however, is contentious. The YXXΦ and LL motifs conserved in the NAS family have been linked to maintenance of enzyme structure, and vesicular localization and movement (Nozoye et al., 2014a). Studies of various NAS homologs have yielded conflicting results. For example, vesicular localization have only been confirmed in OsNAS2, ZmNAS1 and ZmNAS2, while ZmNAS3 and the AtNAS family, localized to the cytoplasm (Mizuno et al., 2003; Nozoye et al., 2014a,b). It was proposed by Nozoye et al. (2014b) that vesicular localization is required for DMA synthesis, hence its specificity to the graminaceous homologs. With chickpea lacking in that regard, its localization pattern is likely to be more similar to that of AtNAS, though more studies are required to confirm this. ### Constitutive Expression of CaNAS2 and GmFER Does not Increase Seed Fe Concentration but Is Likely to Increase Seed Fe Bioavailability As demonstrated in this study, constitutive overexpression of the endogenous CaNAS2 gene combined with constitutive expression of the GmFER gene in chickpea resulted in higher leaf Fe in one event, and lower leaf Zn in the other event, with no apparent effects on yield or morphology in either event. Seed Fe, Zn and Mn concentrations were not changed in either event. These results suggest that high expression of the CaNAS2 and GmFer transgenes is not an effective strategy for improving the micronutrient composition of chickpea grain. Potentially a better strategy in the future would be to use a seed-specific promoter to drive GmFer expression in conjunction with CaNAS2 overexpression. Indeed, this method has been demonstrated to be extremely effective in rice, producing a 7.5 fold increase in the Fe concentration of polished seeds with no yield penalty (Trijatmiko et al., 2016). Due to high levels of inhibitory compounds (i.e., phytic acid), the Fe bioavailability in pulses is low relative to other crops (Hemalatha et al., 2007). As NA is a known promoter of Fe bioavailability, doubling the concentration of NA in chickpea flour may increase Fe bioavailability without alterations to seed mineral concentration (Zheng et al., 2010; Eagling et al., 2014). Future in vitro Fe bioavailability studies utilizing the Caco-2 cell line assay are needed to confirm increased Fe bioavailability in high-NA chickpea events. ### AUTHOR CONTRIBUTIONS SM, BW, SD, and AJ conceived and designed the project. GT, SD, HL, AC, TH, MK, AJ, JPB, and JTB designed and performed the experiments. GT, JTB, and JPB data analysis. GT, JTB, JPB, and AJ wrote the paper. ### ACKNOWLEDGMENTS We would like to thank the Australian Government for the funding through the Tropical Pulses for Queensland project. We also thank the following people: Dr TJ Higgins for his invaluable guidance on chickpea transformation, Queensland Department of Agriculture and Fisheries, and Dr Yash Chauhan for provision of seed material, Waite Analytical Services, Dr Bulukani Mlalazi, Dr Charlotte Allen, and Miss Karine Moromizato for their guidance and assistance with trace elemental analyses. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00788/full#supplementary-material ## REFERENCES collection of Italian durum wheat cultivars. Field Crops Res. 111, 235–242. doi: 10.1016/j.fcr.2008.12.010 metabolite levels during the iron deficiency response of rice. Rice 10:14. doi: 10.1186/s12284-017-0152-7 Conflict of Interest Statement: AJ is an editor for the Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges topic of Frontiers in Plant Science. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Tan, Das Bhowmik, Hoang, Karbaschi, Long, Cheng, Bonneau, Beasley, Johnson, Williams and Mundree. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Biofortification of Cereals With Foliar Selenium and Iodine Could Reduce Hypothyroidism ### Graham Lyons\* School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA, Australia Concurrent selenium and iodine deficiencies are widespread, in both developing and developed countries. Salt iodisation is insufficient to ensure global iodine adequacy, with an estimated one-third of humanity at risk of hypothyroidism and associated iodine deficiency disorders (IDD). Agronomic biofortification of food crops, especially staples such as cereals, which are consumed widely, may be an effective component of a food system strategy to reduce selenium and iodine malnutrition. Iodine and selenium are needed in the optimum intake range for thyroid health, hence joint biofortification makes sense for areas deficient in both. Foliar application is recommended as the most effective, efficient, least wasteful method for selenium and iodine biofortification. Currently, selenium is easier to increase in grain, fruit, and storage roots by this method, being more phloem mobile than iodine. Nevertheless, strategic timing (around heading is usually best), use of surfactants and co-application with potassium nitrate can increase the effectiveness of foliar iodine biofortification. More research is needed on iodine transporters and iodine volatilisation in plants, bioavailability of iodine in biofortified plant products, and roles for nano selenium and iodine in biofortification. For adoption, farmers need an incentive such as access to a premium functional food market, a subsidy or increased grain yield resulting from possible synergies with co-applied fertilisers, enhancers, fungicides, and insecticides. Further research is needed to inform these aspects of foliar agronomic biofortification. Keywords: biofortification, cereals, deficiency, hypothyroidism, iodine, iodine deficiency disorders (IDD), ### INTRODUCTION selenium, wheat Malnutrition is the main cause of global human mortality, with over 50% of deaths attributed to diet-related diseases. Micronutrient deficiencies, notably iron (Fe), zinc (Zn), selenium (Se), iodine (I), and certain vitamins are widespread globally, affecting about 60% of the world's population, and in many areas multiple deficiencies occur (Lyons and Cakmak, 2012). Dysfunctional food systems fail to provide optimum nutrition to populations, especially to vulnerable sub-groups such as infants, children, and pregnant and nursing women (White and Broadley, 2009). This has been exacerbated by high-yielding Green Revolution cereal varieties with grain often less micronutrient-dense than previously (Smolen et al., 2016a). #### Edited by: Alexander Arthur Theodore Johnson, University of Melbourne, Australia #### Reviewed by: Elizabeth Pilon-Smits, Colorado State University, United States Michael A. Grusak, USDA-ARS Children's Nutrition Research Center, United States \Correspondence: Graham Lyons [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 18 January 2018 Accepted: 15 May 2018 Published: 08 June 2018 #### Citation: Lyons G (2018) Biofortification of Cereals With Foliar Selenium and Iodine Could Reduce Hypothyroidism. Front. Plant Sci. 9:730. doi: 10.3389/fpls.2018.00730 132* Biofortification of staple crops to achieve higher micronutrient concentrations in edible parts represents a food system strategy to address dietary deficiencies, with the potential to reach the neediest of the population (Haug et al., 2007; Bouis and Welch, 2010; Lyons and Cakmak, 2012). This approach, which links a nutritious agriculture with human health, can be more effective and sustainable than provision of food supplements (Lyons, 2014). Previous research suggests that genetic biofortification (plant breeding and genetic engineering) may be more suitable for increasing pro-vitamin A carotenoids and Fe, whereas an agronomic (fertiliser) strategy may be more effective for Zn, Se, and I (Genc et al., 2004; Cakmak, 2008; Bouis and Welch, 2010; Lyons and Cakmak, 2012). Transgenics may play an important role in micronutrient biofortification (White and Broadley, 2009), as shown by the high-Fe variant of the popular IR64 rice variety (Trijatmiko et al., 2016). Biofortification using conventional breeding or transgenics is a long-term process. Furthermore, the success of genetic biofortification of Se and I depends largely on their plant available concentrations in the soil solution. In most soils, plant available Se, for example, comprises only about 2.5% of total Se (Tan et al., 2002). Agronomic and genetic biofortification are hence complementary (White and Broadley, 2009; Lyons and Cakmak, 2012). If minerals such as Fe, Zn, Se, and I can be increased in staple foods, population status of these minerals can be increased without behavioural change (Bouis and Welch, 2010). Hence widely consumed cereals, especially wheat, provide a suitable vehicle for increasing population Se status using agronomic biofortification (Broadley et al., 2006; White and Broadley, 2009; Fairweather-Tait et al., 2011). The iodothyronine deiodinases D1, D2, and D3, which are selenoenzymes, control thyroid hormone turnover and hence are crucial in thyroid gland metabolism. Selenium supply is prioritised to the thyroid under conditions of Se restriction. Concurrent deficiencies of Se and I may exacerbate hypothyroidism (Schomburg and Kohrle, 2008; Fairweather-Tait et al., 2011; Kohrle, 2013; Gashu et al., 2016), and low Se status increases risk of goitre, especially in women (Rasmussen et al., 2011; Schomburg, 2012; Wu et al., 2015). The more severe the Se deficiency, the less effective is I supplementation in alleviating goitre (Zimmermann et al., 2000; Drutel et al., 2013; O'Kane et al., 2018). Moreover, Se-dependent glutathione peroxidases protect the thyroid against oxidative stress, for example, due to excess I (Schomburg and Kohrle, 2008; Schomburg, 2012; Drutel et al., 2013; Kohrle, 2013). Hypothyroidism is not the only pathological condition that can be exacerbated by concurrent I and Se deficiencies: myxoedematous cretinism, whose aetiology requires I and Se deficiency accompanied by a goitrogen (for example, TGF-beta, thiocyanates from cassava, Fusarium toxins in wheat), exists in parts of Tibet and the Democratic Republic of Congo (Contempre et al., 1992; Schomburg and Kohrle, 2008; Christophersen et al., 2012; Kohrle, 2013). In myxoedematous cretinism, hypothyroidism persists despite I supplementation (Contempre et al., 1992). Where both deficiencies occur, it is important to normalise I intake and status first, before supplementing with Se. If Se is supplemented first, hypothyroidism can worsen in the short term (Contempre et al., 1992). This mini-review will focus on research on agronomic biofortification of cereals with Se and I, and explore the proposal that simultaneous application of these micronutrients has the potential to reduce hypothyroidism and related iodine deficiency disorders (IDD) in areas with concurrent Se and I deficiencies (Figure 1). ### SELENIUM ### Profound Influence on Human Health With a Variable Distribution The importance of Se to human health, in terms of its key roles in the thyroid, brain, heart, and gonads, along with heavy metal-binding, antioxidant, anti-cancer, anti-bacterial, and antiviral activity, is indicated by its status as the only micronutrient to be specified in the human genome, as selenocysteine, the twenty-first amino acid (Rayman, 2000, 2002). Its deficiency is also linked to several diseases, including the osteoarthropathy, Kashin-Beck disease (KBD), which is still prevalent in parts of China, including the Loess Plateau in Shaanxi Province, and Tibet. Aetiological factors for KBD include Fusarium mycotoxins in infected grain, organic acids in drinking water, low dietary Se, and gene polymorphisms (Fairweather-Tait et al., 2011; Bissardon et al., 2017). Although much less common than Se deficiency, Se toxicity can occur, for example in Enshi in the Chinese province of Hubei, when selenosis, characterised by hair loss and thickened nails, occurred, particularly from 1961 to 1964. It was caused by eating crops grown on high-Se soil (Yang et al., 1983). Daily recommended intake of Se is mostly 40–75 µg/day globally, with <30 µg/day inadequate and >900 µg/day potentially harmful; however, tolerable upper limits have been set lower, in the range of 400–450 µg/day for the United Kingdom, United States, Canada, EU, Australia, and New Zealand (Fairweather-Tait et al., 2011). Selenium's interplay with human physiology is complex and integral, and deficiency, sufficiency, and toxicity span a relatively narrow range of Se intake and status (Vinceti et al., 2009; Fairweather-Tait et al., 2011; Winkel et al., 2012). Selenium delivery in a food system depends mainly on the levels of plant available Se in soils used for agriculture. Selenium is ubiquitous but of uneven plant-availability, hence its variability in populations and their sub-groups. It is estimated that up to a billion people are deficient in Se (Combs, 2001; Haug et al., 2007; Winkel et al., 2012; Ros et al., 2016). The element's availability in soils depends on soil pH, redox potential, cation exchange capacity, and levels of Fe, sulphur, aluminium, and carbon (Broadley et al., 2006; Chilimba et al., 2011; Christophersen et al., 2012; Winkel et al., 2012). ### Agronomic Biofortification: Foliar Selenate More Efficient Selenium is well suited to agronomic biofortification of food crops. In the selenate form, it is readily taken up by plants growing on most soils, then transported throughout the plant, accumulating in edible parts. In cereals, it is converted mostly into selenomethionine, which is well represented in grain endosperm, hence Se can be abundant and bioavailable in milled products such as white flour and polished rice (Lyons and Cakmak, 2012). Selenium form is important for effective biofortification. Most studies have shown selenate (where Se exists in its highest oxidation state, +6) to be easily the most effective form when applied to the soil and usually more effective than selenite (Se +4) when applied as a foliar (Broadley et al., 2006; Boldrin et al., 2013; Mao et al., 2014; Ros et al., 2016; Xiong et al., 2018). A recent review found selenate to be 33 times more effective overall than selenite (Ros et al., 2016). In many soils, selenite is rapidly adsorbed on clay colloids, rendering it poorly available to plants. Dry climate, low organic matter, high temperature, high soil pH, and aeration are likely to increase the selenate: selenite ratio in the soil and hence the availability of Se to plants (Christophersen et al., 2012). In Finland, the use of Se (selenate) fertilisers commenced on a national scale in 1984, resulting in a fourfold increase in dietary Se intake and doubling of the plasma/serum Se concentrations of the study population. There were concerns that the addition of Se in this manner may have long-term environmental effects. In California, for example, drainage water collected from an irrigated area overlaying a high-Se shale resulted in deaths and malformations in fish and aquatic birds at the Kesterson reservoir in the 1980s (Hartikainen, 2005). A study of lake and ground water in Finland in 1992 found no differences in Se concentration in water from lakes in agricultural and nonagricultural areas. Ground water samples were variable in Se (33–260 µg/l), partly explained by different Se concentrations of bedrock and sediments, and some leaching from fertilisers (as indicated by correlations with phosphorus and nitrogen) in certain areas (Mäkelä et al., 1995). The ongoing Finnish biofortification programme demonstrates the relative safety, effectiveness, ease, and cost-efficiency of this strategy (Eurola et al., 1990; Broadley et al., 2006; Haug et al., 2007; Winkel et al., 2012; Ros et al., 2016). This model could be applied to other low-Se countries, like Malawi (Chilimba et al., 2012). Nevertheless, Se soil biofortification is a relatively wasteful process. The recovery of soil-applied Se in wheat grain varies from 5 to 32%, with an estimated average of about 12% (Eich-Greatorex et al., 2007; Haug et al., 2007; Broadley et al., 2010; Lyons and Cakmak, 2012; Ros et al., 2016). Selenium is a valuable, mostly non-renewable resource, which should be conserved (Haug et al., 2007; Ros et al., 2016). Foliar application has usually been found to be more efficient than soil application for Se (Ylaranta, 1984; Mao et al., 2014; Winkel et al., 2015; Ros et al., 2016; Gupta and Gupta, 2017). Foliar application not only obviates the soil factors that can reduce the effectiveness of soil agronomic biofortification of Se, but also reduces possible environmental Se accumulation (see above) as less Se is applied per hectare. Timing of foliar Se and I application is important, with the best effect usually obtained between booting and early milk stage, with heading, when green leaf cover is maximised, the "best bet" for an effective single application. A recent meta-analysis enables estimation of the amount of selenate needed to increase grain Se from 7 to 100 µg/kg: 30–60 g/ha soil-applied selenate, and 4.5–10 g/ha foliar selenate. This study found foliar-applied Se to be on average eight times more efficient than soil-applied Se (Ros et al., 2016). In tropical/sub-tropical countries where Se fertilisers are unavailable, leaves of the Drumstick tree (Moringa oleifera), which has exceptional ability to take up and accumulate Se, can provide useful levels of Se, even when grown on soils that provide little Se to most other plants (Lyons et al., 2015). ### IODINE ### Iodised Salt Needs Help to Fix Global Iodine Insufficiency Iodine is essential to humans, being required for synthesis of thyroid hormones, which are essential for human development and health. Requirement is in the range 90–250 µg/day. Inadequate I is one of the major micronutrient deficiencies, leading to a range of clinical and social issues known as IDD. The classic symptom of I deficiency is an enlarged thyroid, known as goitre (Zimmermann et al., 2008). The safe upper limit of I intake is estimated at 1000–1100 µg/day; chronic intakes above this level can increase risk of Graves disease (Surks et al., 2004; Leung et al., 2015). Like Se, plant-available I is unevenly distributed (Figure 1): the sea is an important source, hence I in food systems usually declines with distance from it. Inland, high rainfall, mountainous areas are notoriously deficient in I. The overall global average soil I concentration is 2.6 mg/kg (Hetzel, 1989; Watts et al., 2010), but I concentration in plants grown on I-deficient soils may be as low as 10 µg/kg, compared with 1 mg/kg in plants on an I-replete soil (Hetzel and Pandav, 1994). Although the number of countries designated as I deficient halved in the decade to 2014 (Gonzali et al., 2017), I deficiency remains prevalent, affecting an estimated 33% of humanity (Fuge and Johnson, 2015). Marginal I status is even present in developed countries, including England, Germany, Italy, and Australia (Andersson et al., 2012). It is apparent that iodisation of salt is insufficient to ensure overall I adequacy. Contributing factors include lack of availability of iodised salt for all households, food manufacturers not using iodised salt, volatilisation of I during food transport, storage, and cooking (on average, 20% of I in iodised salt is lost during cooking), and in many countries salt consumption has declined due to public health measures to reduce hypertension (Winger et al., 2008; White and Broadley, 2009; Comandini et al., 2013; Medrano-Macias et al., 2016; Smolen et al., 2016a; Cakmak et al., 2017). Most terrestrial foods are low in I. Strategies complementary to the iodised salt programme are needed, such as production of I-rich plants (White and Broadley, 2009; Comandini et al., 2013; Medrano-Macias et al., 2016; Smolen et al., 2016a; Cakmak et al., 2017). Vegetables biofortified with foliar I showed a high I stability during cooking (Comandini et al., 2013). Genetic approaches may be productive, for example metabolic engineering to reduce the problem of I volatilisation (Gonzali et al., 2017). ### Agronomic Biofortification: Foliar Iodate More Effective, but Easier to Biofortify Leaves Than Fruits, Roots, Grains, and Seeds To address I insufficiency, researchers have urged the WHO to move beyond an iodised salt focus to a broader food system strategy that includes I biofortification of a range of vegetables (Smolen et al., 2016a). A case study of introducing I via agriculture was a spectacular success in Xinjiang province in north-west China. Potassium iodate was dripped into irrigation canals and resulted in a threefold increase in soil I levels, a twofold increase in I in wheat straw, increases in animal and poultry production, and in humans a 50% reduction in infant mortality and virtual elimination of IDD. Benefits were evident up to 7 years later (Jiang et al., 1997; Duxbury et al., 2015). Most studies have shown that iodate is more suitable than iodide for biofortification (Mackowiak and Grossl, 1999; Dai et al., 2006 Lawson et al., 2015; Medrano-Macias et al., 2016; Smolen et al., 2016b; Cakmak et al., 2017). Iodate is also more likely than iodide to promote plant growth and less likely to be phytotoxic (Borst-Pauwels, 1961; Blasco et al., 2008). Iodide is more available than iodate in solution culture, while under field conditions it is more subject to cumulative losses (Lawson et al., 2015). Iodine in plants, unlike Se, is transported mostly (but not entirely: see below) in xylem tissue (Mackowiak and Grossl, 1999), hence it is relatively easy to biofortify leaves, and thus leafy vegetables such as cabbage, lettuce, spinach (Smolen et al., 2014, 2016a,b). It is more difficult to increase I levels in grain or storage roots/tubers (Mackowiak and Grossl, 1999; Hurtevent et al., 2013; Mao et al., 2014; Medrano-Macias et al., 2016; Gonzali et al., 2017). Hence there are more published articles to date on I biofortification of vegetables than for cereals. These vegetable articles provide valuable knowledge of I behaviour in plants that can be applied to cereals. ## Evidence for Phloem Mobility Supports Iodine Biofortification for Cereals A comprehensive study that included glasshouse and field trials of cereals (wheat, rice, maize) in Pakistan, Brazil, Thailand, and Turkey, showed that foliar-applied I can increase grain I (Cakmak et al., 2017). For example, in a pot trial, wheat grain I was increased from 21 to 296 µg/kg using two applications (at heading and early milk stage) of potassium iodate (0.065%) plus a non-ionic surfactant (0.05%) and potassium nitrate (1%). The surfactant and potassium nitrate had an additive effect in enhancing I biofortification. In a field trial in Brazil, potassium iodate (0.05%) applied twice increased grain I from 8 to 485 µg/kg. Other studies also found that surfactants increased the efficiency of foliar micronutrient biofortification (Lawson et al., 2015; Gonzali et al., 2017). Foliar I biofortification was most effective for wheat, followed by rice, then maize (Cakmak et al., 2017). The study of Cakmak et al. (2017) adds to recent evidence of phloem mobility of I in wheat (Hurtevent et al., 2013) and vegetables (Kiferle et al., 2013; Smolen et al., 2014; Li et al., 2017). The mechanism of potassium nitrate's enhancement of leaf absorption and possibly translocation to grain of I may relate to the chemical similarity of nitrate and iodate and is worthy of investigation (Cakmak et al., 2017). ### BIOFORTIFICATION OF CEREALS WITH SELENIUM AND IODINE COULD REDUCE IODINE DEFICIENCY DISORDERS ### Combined Selenium and Iodine Foliar Biofortification: A Promising Strategy for Many Areas In the extensive parts of Sub Saharan Africa, China, South America, Europe, and New Zealand with concurrent Se and I deficiencies (Figure 1) (Hetzel and Pandav, 1994; Oldfield, 1999; Gashu et al., 2016), foliar agronomic biofortification with both Se and I may be effective in increasing the supply of both micronutrients in food systems (Smolen et al., 2016a). Resulting health benefits would be likely to include reduced incidence and prevalence of hypothyroidism with its consequent spectrum of IDD and myxoedematous cretinism. The suitability of foliar Se application for cereal grain biofortification, irrespective of soil type, was discussed above, while the findings of Cakmak et al. (2017) for foliar I biofortification of cereals are promising. Given the observed enhancement of I biofortification provided by potassium nitrate, trials to assess its effect on Se foliar biofortification may also be fruitful. In view of the optimum molar ratio of I:Se, which is in the range of 4.4–8.8:1 (with an average around 6) in the human diet, calculated from the RDIs of 150–250 µg/day for I and 55– 65 µg/day for Se (Smolen et al., 2016a), plausible target levels of I and Se in cereal grain could be 1.0 and 0.25 mg/kg, respectively. Toxic effects can be expected at chronic Se intakes in livestock feed/human food that exceed 1 mg/kg (Hartikainen, 2005). There is an agreeable symmetry in a joint biofortification concept for Se and I, their importance for the thyroid notwithstanding, given their juxtaposition on the Periodic Table. ### Could Se+I Foliar Biofortification of Cereals Be Attractive to Farmers? For agronomic biofortification to become commercial, it needs to benefit both producers and consumers (Bouis and Welch, 2010; Cakmak et al., 2010). Cereal yield is unlikely to be increased by Se and/or I application (Lyons and Cakmak, 2012; Ros et al., 2016), therefore fertiliser containing Se and I may need to be subsidised (Chilimba et al., 2012), or a biofortified product could attract a premium price as a desirable functional food. Although considered to be non-essential to plants, Se and I can be beneficial. For example, Se addition increased biomass in mungbean (Phaseolus aureus) (Malik et al., 2010) and turnip (Brassica rapa var. rapa) (Xiong et al., 2018), increased seed production in canola (Brassica rapa) (Lyons et al., 2009), and improved quality and shelf-life of vegetables and fruits (Puccinelli et al., 2017). Selenate and selenite at low doses increased growth and sulphur accumulation in wheat seedlings, but these effects were not seen in grain (Boldrin et al., 2016). Iodine use in agriculture has been reviewed by Medrano-Macias et al. (2016). Iodine is involved in various plant physiological and biochemical processes (Gonzali et al., 2017). Benefits include growth enhancement, increased nitrogen uptake, increased sugars and amino acids, improved seed viability, and increased tolerance to salinity and heavy metals via induction of antioxidants including ascorbate, glutathione, and superoxide dismutase (Borst-Pauwels, 1961; Medrano-Macias et al., 2016; Gonzali et al., 2017). Potential benefits from applying Se and I, including increased growth and product quality, together with the convenience and economy of combining them with strategic fertiliser, fungicide and insecticide applications, could make Se+I biofortification commercially viable for farmers. ### Further Research Needed Research needed on combined Se+I biofortification includes evaluation of potential enhancers, including salicylic acid, a phytohormone-like compound, which improved tomato fruit biofortification with I (Smolen et al., 2015), the pineal gland hormone melatonin, which is also present in plants and can act as a synergist with antifungal agents (Zhang et al., 2017), the carrier dimethyl sulfoxide, which increased the effectiveness of foliar Fe application (Leonard, 2006) and synergists such as potassium nitrate (Cakmak et al., 2017). Trials of co-application with fungicides and insecticides are also recommended, due to promising earlier findings (Mahmoud et al., 1996; Costa et al., 2003; Zhang et al., 2003; Hanson et al., 2004). Knowledge of I transporters in plants is incomplete (White and Broadley, 2009; Gonzali et al., 2017), and such research could include I volatilisation studies (Gonzali et al., 2017). An efficient single application of Se+I will be more acceptable to farmers than multiple applications. Nanotechnologies in agriculture are attracting interest (De Rosa et al., 2010; Liu and Lal, 2015). Bioavailable biogenic elemental Se (BioSe), for example, is widespread in the microbial environment (Winkel et al., 2012). For roles in foliar biofortification, Se and I nanoparticles need to be well characterised, including particle size: stomatal openings are about 20 nm in diameter, thus movement of particles larger than this is problematic (Alshaal and El-Ramady, 2017). More bioavailability studies that examine losses of Se and I from biofortified cereals during milling and during various cooking methods are also required, along with speciation of I in biofortified cereals. ### REFERENCES ### AUTHOR CONTRIBUTIONS GL researched, wrote, and checked the manuscript. ### ACKNOWLEDGMENTS This article was written while the author was working on the project Field Testing of Sodicity- and Salinity-Tolerant Oat Varieties, supported by the South Australian Grain Industry Trust Fund (SAGIT). The author and colleagues' earlier agronomic biofortification field trials using selenium and iodine were supported by HarvestPlus, The Grains Research and Development Corporation (Australia), The University of Adelaide, SAGIT, Northwest A&F University (Shaanxi, China), and the International Centre for Tropical Agriculture (CIAT, Cali, Colombia). This article is dedicated to Drs. Robin Graham, Ross Welch, and Howarth Bouis, the founders of HarvestPlus. Dr. Bouis was awarded jointly the 2016 World Food Prize. through fertilizer strategy. Plant Soil 418, 319–335. doi: 10.1007/s11104-017- 3295-9 fpls-09-00730 June 6, 2018 Time: 16:18 # 6 and K. Moran (Norcross, GA: International Plant Nutrition Institute), 97–122. fpls-09-00730 June 6, 2018 Time: 16:18 # 7 Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Lyons. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. fpls-09-00730 June 6, 2018 Time: 16:18 # 8 # Should Heavy Metals Be Monitored in Foods Derived From Soils Fertilized With Animal Waste? Rafael da Rosa Couto<sup>1</sup> \, Jucinei J. Comin<sup>2</sup> , Monique Souza<sup>2</sup> , Felipe K. Ricachenevsky 3,4 , Marcos A. Lana5,6, Luciano C. Gatiboni <sup>7</sup> , Carlos A. Ceretta<sup>8</sup> and Gustavo Brunetto<sup>8</sup> <sup>1</sup> Técnico em Agroecologia, Instituto Federal Catarinense, Rio do Sul, Brazil, <sup>2</sup> Agroecossistemas, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, Brazil, <sup>3</sup> Departamento de Biologia, Agrobiologia, Universidade Federal de Santa Maria, Santa Maria, Brazil, <sup>4</sup> Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, <sup>5</sup> Department of Crop Production Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden, <sup>6</sup> SUSLAND, Leibniz Centre for Agricultural Landscape Research, Müncheberg, Germany, <sup>7</sup> Ciência do Solo, Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, Brazil, <sup>8</sup> Ciência do Solo, Departamento de Solos, Universidade Federal de Santa Maria, Santa Maria, Brazil Keywords: heavy metals, animal waste, soil fertilization, residue management, seed contamination #### Edited by: Jose M. Garcia-Mina, Universidad de Navarra, Spain #### Reviewed by: Heitor Cantarella, Instituto Agronômico de Campinas (IAC), Brazil Lourdes Hernandez-Apaolaza, Universidad Autonoma de Madrid, Spain \Correspondence: Rafael da Rosa Couto [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 21 February 2018 Accepted: 15 May 2018 Published: 05 June 2018 #### Citation: da Rosa Couto R, Comin JJ, Souza M, Ricachenevsky FK, Lana MA, Gatiboni LC, Ceretta CA and Brunetto G (2018) Should Heavy Metals Be Monitored in Foods Derived From Soils Fertilized With Animal Waste? Front. Plant Sci. 9:732. doi: 10.3389/fpls.2018.00732 Heavy metals (HM) represent a large group of elements with atomic density >5 g cm−<sup>3</sup> or atomic number >20 (Saidur et al., 2017), among which some are essential to plants, such as iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), and manganese (Mn). However, HMs may be contaminants and/or pollutants, depending on the concentration in soils. HMs such as Cu, Zn, Ni, and chromium (Cr) are essential to human beings, and biofortification approaches to improve levels of some elements in plant edible parts are underway (Bouis et al., 2012; Ricachenevsky et al., 2015). However, these HMs may be toxic if accumulated, and may only be ingested in very small quantities (EPA-U.S. Environmental Protection Agency, 1995; FAO-Food Agriculture Organization of the United Nations, 1995; Tchounwou et al., 2012). On the other hand, Pb, Cd, As, and Br are not essential and can be toxic even at low concentrations (Tchounwou et al., 2012). The safe daily intake level for As, Cd, Cr, Cu Ni, Pb, and Zn is 20, 300, 1500, 4, 20, 40, 300 µg kg−<sup>1</sup> of body weight per day, respectively (EPA-U.S. Environmental Protection Agency, 1993). These levels are based on the degree to which the element may cause disturbance, the capacity of the body to accumulate the element and the weight of the individual who is ingesting it (Abbasi et al., 2013). However, when HMs are ingested for long periods, even at doses considered safe, they can cause harmful effects, known as chronic intoxication (Jorge Mendoza et al., 2017; Li et al., 2017). The increase in total HM concentration and their chemical forms in soils can occur naturally due to atmospheric deposition, weathering of rocks, and anthropic activities such as mining, deposition of ash from coal burning, application of pesticides in plants, addition of mineral and organic fertilizers, among others (Guilherme et al., 2005). HM accumulation in the soil is typically assessed by indicators such as Geo-accumulation index (Igeo) (Equation 1) (Müller, 1979) and Enrichment Factor (EF) (Equation 2) (Abbasi et al., 2013) that allow the identification of the presence and the intensity of deposition of anthropogenic contaminants in topsoil. $$Age = \log\_2\left(\frac{\lfloor \frac{\lfloor C\_{\rm u} \rfloor}{1.5} \* \lfloor B\_{\rm u} \rfloor}{}\_{\ast}\right) \tag{1}$$ where: Cn is the measured concentration in the soil for the metal n, Bn is the background value for the metal n, and the factor 1.5 is used because of possible variations of the background data due to lithological variations. $$EF = \frac{\left[\frac{metal}{RE}\right]sample}{\left[\frac{metal}{RE}\right]control} \tag{2}$$ where: RE is the value of metal, adopted as Reference Element. High HM concentrations in soils may cause intoxication upon inhalation, contact with the skin, indirect ingestion of soil and intake of fruits, vegetables, grains, and their byproducts (Zheng et al., 2010; Chabukdhara and Nema, 2013; Chen et al., 2016; Jiang et al., 2017). Plants grown in soils contaminated/polluted with HM tend to absorb, accumulate, transport, and redistribute larger amounts of HM. This is likely due to the presence of nonselective essential element transporters. For instance, iron high affinity transporter IRT1 of the model plant Arabidopsis thaliana, which is necessary for Fe acquisition under iron deficiency, is known to also transport Zn, Mn, Ni, Co, and Cd, possibly leading to metal toxicity under Fe deficiency (Korshunova et al., 1999; Barberon et al., 2014; Ricachenevsky et al., 2018). In rice, IRT1 might also transport Zn and Cd (Lee and An, 2009). Arsenic uptake is also performed by phosphate transporters (as arsenate) or by silicon transporters (as arsenite), which are not able to distinguish between these elements (Kochian et al., 2015). Thus, non-selective transport leads to accumulation of toxic elements, which might end up accumulating in grains or other harvested parts, and may change nutrient abundance and distribution (Punshon et al., 2018). These agricultural products containing high HM concentration might then be used for human consumption directly or indirectly through the intake of processed foods (Hariri et al., 2015; Avkopashvili et al., 2017). To assess the risk of ingestion of a particular HM over the life of an individual, it is necessary to consider the period of ingestion. Therefore, indexes have been established to verify the risk that certain elements, such as HMs, could cause to human beings (Abbasi et al., 2013). A few examples of these indexes are the Health Risk Index (HRI), Target Hazard Quotient (THQ) and Target Cancer Risk (TCR) (Equation 5) (EPA-U.S. Environmental Protection Agency, 2010). $$HRI = \frac{(C\_n \ltimes D\_n)}{\left(R \lhd D \lhd B \, W\right)}\tag{3}$$ where: Cn, total concentration of the metal in edible plant organ (mg kg−<sup>1</sup> ); Dn, daily intake (g day−<sup>1</sup> ); BW, average body weight (kg); RfD, reference dose (EPA-U.S. Environmental Protection Agency, 2010). $$HQ = \frac{\left(C\_n \times D\_n \times 10^{-3} \,\mathrm{\chi EF\_r \times ED\_{tot}}\right)}{RfD \mathrm{xB}W\_a \mathrm{xAT\_n}}\tag{4}$$ where: EF<sup>r</sup> , exposure frequency (days); EDtot, exposure duration (years); ATn, average exposure time to non-carcinogenic heavy metals (e.g., EDtot x 365 days/year). $$TCR = \frac{\left(C\_n \varkappa D\_n \varkappa 10^{-3} \text{\AA} \text{\textdegree C} \text{\textdegree S}\_0 \text{\textdegree E} F\_r \text{\textdegree} D\_{tot}\right)}{\left(BW\_a \varkappa A T\_n\right)} \tag{5}$$ where: CPS0, carcinogenic potential (µg g−<sup>1</sup> day−<sup>1</sup> ). The effects of HM accumulation in soil, excess uptake by plants, and the risks that HM-contaminated foods can promote to human beings are commonly reported in mining regions (Qing et al., 2015; de Souza et al., 2017; Li et al., 2017). As example, the release and drifting of dust from coal mines in the Qingshui River basin (China) has resulted in pollution of arable soils. Despite the knowledge associated to the deposition of HMs, few studies approach the increase of HM concentration in different edible plant organs cultivated on soils subjected to a long history of animal waste application. Different environmental agencies have established acceptable levels of HM in food. FAO and EPA-USA established maximum levels for Cu, Zn, Cd, Pb, Cr, and Ni in crop grains of 20, 50, 0.1, 0.2, 1, and 0.04, respectively. However, studies on soils subjected to the addition of urban sludge and animal residues reported increased HM concentration above these limits in grains, fruits, and vegetables (Suarez-Tapia et al., 2017; Zhang et al., 2017). The use of wastewater for irrigation in Iran containing 0.06, 0.010, 0.01, 0.010, and 0.010 mg kg−<sup>1</sup> of Cu, Zn, Cd, Pb, Cr, and Ni, respectively, caused the accumulation of Cd, Cr, and Pb in wheat and corn grains above the limits established by the EPA. Health risks to adults and especially children by Cu, Cd, and Cr intake in corn and wheat grains were also reported (Asgari and Cornelis, 2015). Animal waste contains HM derived from drugs or feed (Gunkel-Grillon et al., 2015; Couto et al., 2016). It is worth mentioning that soils with frequent application of organic wastes typically have higher HM concentrations than those described in studies where negative effects of excess HM on edible plant organs and human health risk have been reported, indicating that we might be underestimating the contamination of foods derived from such areas (Table 1). Studies that address the effects of increasing HM concentration in soils subjected to long-term animal waste application and consequent changes of HM concentration in edible plant organs are still scarce. Although organic fertilization recommendations exist both for conventional and organic production systems, the application of organic residues is often carried out indiscriminately in regard to HMs content, increasing their concentration in soils and likely increasing of HM concentration in edible plant organs (Couto et al., 2016; Suarez-Tapia et al., 2017; Zhang et al., 2017). A very important aspect is that in organic production systems, organic residues (including animal manure) are the main—if not the only—source of nutrients for the crops. Considering that organic production systems currently occupy 42 million hectares worldwide (FIBL, 2017), with a global growth rate of 4.5% per year, the risk of HM contamination in the food systems is present, especially in some regions of the world. In Brazil, Japan, and the European Union, the growth rate of the area cultivated under the organic system is 30, 13, and 8% per year, respectively (FIBL, 2017). This emergent risk indicates that the organic residues that will be used as source of nutrients for the crops needs to be assessed in terms of HM concentration. In Brazil, the applications of pig slurry, cattle slurry, and pig deep litter for 10 years in sandy soil with low organic matter content under no-till increased Ni, Cu, and Zn concentrations in shoots and grains of corn and wheat (da Rosa Couto et al., 2018). TABLE 1 \| Heavy metal contents in soils and vegetables of diverse areas of the world and risks to human health. The applications of organic wastes (pig slurry, cattle slurry, and pig deep litter) and mineral fertilizers also increased the values of HRI and THQ for Br and Zn, presenting health risks to adults and especially children who have lower body weight (da Rosa Couto et al., 2018). They also report that Cu concentrations in corn grains of plants grown in soil with application of pig deep litter and cattle slurry were 2.7 and 2.2 mg Cu kg−<sup>1</sup> , respectively. On the other hand, Zn concentrations in corn grains of plants grown in soil with application of pig deep litter, pig slurry, and cattle slurry were 26, 31, and 23 mg Zn kg−<sup>1</sup> , respectively. In the grains of wheat grown in soil with the application of pig deep litter, pig slurry, and cattle slurry, concentrations of Cu were 6.0, 6.0, and 4.5 mg kg−<sup>1</sup> , respectively, and Zn were 96, 95, and 84 mg kg−<sup>1</sup> , respectively. Thus, Cu and Zn concentrations in grains of corn and wheat grown in soil with a long history of application of organic wastes were higher than those found in plants grown in the control soil or even with the application of mineral fertilizer. This justifies the monitoring of concentrations of elements in grains of plants grown in soils with a long history of organic waste application, especially in soils with low capacity for adsorption of elements, such as sandy soils with low organic matter (Brunetto et al., 2014). Plants have the potential to absorb and accumulate larger amounts of several heavy metals. In studying heavy metal contents in vegetables fertilized with wastewater in India, Singh et al. (2010) found that the concentrations of Cd in plants varied from >2 to 15 mg kg−<sup>1</sup> , while Pb ranged from >1 to 28 mg kg−<sup>1</sup> and Ni from >1 to 41 mg kg−<sup>1</sup> . The authors verified a risk to consumer health (HRI> 1) through the ingestion of Cd accumulated in radish, cabbage, cauliflower, okra, eggplant wheat and rice; of Pb accumulated in palak, cabbage, cauliflower, Lady's fingers, brinjal, wheat, and rice; and Ni accumulated in palak, cauliflower, wheat, and rice. However, it is important to observe the proportion of vegetables and cereals in the diet, which may change according to the culture of each place and country, causing higher or lower risk. ### REFERENCES ### FINAL CONSIDERATIONS The use of waste as a source of nutrients in plant production systems, traditional, and organic, is common worldwide, and important strategy for nutrient cycling. However, longterm application of such waste causes the increase of HM concentrations in soils, increasing HM uptake by plants and assimilation in edible organs such as grains, as indicated by the data presented in Table 1. As different plant organs can be used in the preparation of numerous products for human consumption, it is necessary to monitor the concentrations of HM in edible plant organs of different species and cultivars fertilized with organic waste. This monitoring can be done through indexes such as Igeo, EF, HRI, THQ, TCR, allowing us to estimate the possible dangers of HMs to the health of children, young adults, and adults who eat food derived from plants grown in soils with a history of animal waste application. Thus, we recommend careful consideration of practices that indiscriminately use animal waste in plant production to avoid HM accumulation and health hazards to consumers. Moreover, strongly indicate that evaluation of metal contamination in foods derived from plants cultivated using animal waste should be commonplace, and further studies of how widespread that is should be conducted by the scientific community. ### AUTHOR CONTRIBUTIONS RdRC: wrote the first draft of the manuscript. MS: organized the database. JC, LG, CC: senior researchers in the field of soil science. They made specific contributions of the area. FR: senior researchers in the field of plant physiology. They made specific contributions of the area. ML: senior researchers in the field of agroecology. They made specific contributions of the area. All authors contributed to the revision of the manuscript, read and approved the version sent. to Improve Human Health: A Scientific Review, eds T. W. Bruulsema, P. Heffer, R. M. Welch, I. Cakmak, K. Moran (Norcross, GA: IPNI), 97–122. health from simultaneous exposure to multiple contaminants in an artisanal gold mine in Serra Pelada, Pará, Brazil. Sci. Tot. Environ. 576, 683–695. doi: 10.1016/j.scitotenv.2016.10.133 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 da Rosa Couto, Comin, Souza, Ricachenevsky, Lana, Gatiboni, Ceretta and Brunetto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Dynamic Modeling of Silicon Bioavailability, Uptake, Transport, and Accumulation: Applicability in Improving the Nutritional Quality of Tomato Mari C. López-Pérez <sup>1</sup> , Fabián Pérez-Labrada<sup>1</sup> , Lino J. Ramírez-Pérez <sup>1</sup> , Antonio Juárez-Maldonado<sup>2</sup> , América B. Morales-Díaz <sup>3</sup> , Susana González-Morales <sup>4</sup> , Luis R. García-Dávila<sup>5</sup> , Jesús García-Mata<sup>6</sup> and Adalberto Benavides-Mendoza<sup>1</sup> \* <sup>1</sup> Departamento de Horticultura, Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico, <sup>2</sup> Departamento de Botánica, Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico, <sup>3</sup> Robótica y Manufactura Avanzada, Centro de Investigación y de Estudios Avanzados Unidad Saltillo, Ramos Arizpe, Mexico, <sup>4</sup> Departamento de Horticultura, CONACYT-Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico, <sup>5</sup> Cosmocel España, Zaragoza, Spain, <sup>6</sup> Cosmocel Brasil, São Paulo, Brazil #### Edited by: Raul Antonio Sperotto, University of Taquari Valley, Brazil #### Reviewed by: Sergio Esposito, University of Naples Federico II, Italy MCarmen Martinez-Ballesta, Consejo Superior de Investigaciones Científicas (CSIC), Spain #### \Correspondence: Adalberto Benavides-Mendoza [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 20 January 2018 Accepted: 27 April 2018 Published: 17 May 2018 #### Citation: López-Pérez MC, Pérez-Labrada F, Ramírez-Pérez LJ, Juárez-Maldonado A, Morales-Díaz AB, González-Morales S, García-Dávila LR, García-Mata J and Benavides-Mendoza A (2018) Dynamic Modeling of Silicon Bioavailability, Uptake, Transport, and Accumulation: Applicability in Improving the Nutritional Quality of Tomato. Front. Plant Sci. 9:647. doi: 10.3389/fpls.2018.00647 Silicon is an essential nutrient for humans, additionally is beneficial for terrestrial plants. In plants Si enhances tolerance to different types of stress; in humans, it improves the metabolism and increases the strength of skeletal and connective tissues as well as of the immune system. Most of the Si intake of humans come from edible plants creating a double benefit: first, because the absorption of Si increases the antioxidants and other phytochemicals in plants, thereby increasing its functional value, and second because the higher concentration of Si in plants increases intake in human consumers. Therefore, it is desirable to raise the availability of Si in the human diet through the agronomic management of Si accumulator species, such as corn, wheat, rice, soybeans, and beans. But also in such species as tomatoes, carrots, and other vegetables, whose per capita consumption has increased. However, there are few systematized recommendations for the application and management of Si fertilizers based on the physicochemical factors that determine their availability, absorption, transport, and deposition in cells and tissues. This study presents updated information about edaphic and plant factors, which determine the absorption, transport, and deposition rates in edible organs. The information was integrated into an estimated dynamic model that approximates the processes previously mentioned in a model that represents a tomato crop in soil and soilless conditions. In the model, on the other hand, was integrated the available information about key environmental factors related to Si absorption and mobilization, such as the temperature, pH, and soil organic matter. The output data of the model were compared against information collected in the literature, finding an adequate adjustment. The use of the model for educational or technical purposes, including the possibility of extending it to other horticultural crops, can increase the understanding of the agronomic management of Si in plants. Keywords: silicates, nutritional quality, stress tolerance, mathematical models, silicon and health ## INTRODUCTION On average a human organism contains 1–2 g of Si, being the third most abundant trace element after Fe and Zn. When it is contained in food in adequate quantity, silicon is effectively absorbed by the human organism (Sripanyakorn et al., 2009), transferring to practically all tissues, but concentrating in greater quantity in the connective tissues (O'Dell and Sunde, 1997; Jugdaohsingh, 2007). With a diet rich in vegetables the daily intake of silicon is between 140 and 204 mg Si day−<sup>1</sup> ; however, in western populations with lower consumption of vegetables, the daily intake can range between 20 and 50 mg day−<sup>1</sup> . Silicon is rarely toxic when taken orally (Arora and Arora, 2017), with a recommended maximum intake of 1,500 mg day−<sup>1</sup> (White and Broadley, 2005). On the other hand, the minimum value of Si consumption to achieve some benefits has been determined at 25 mg day−<sup>1</sup> (Nielsen, 2014). After ingestion, most of the absorbed Si is excreted in the urine (Jugdaohsingh, 2007), most likely as orthosilicic acid and/or magnesium orthosilicate. Plant foods are the primary source of Si in the human diet. This includes grains of cereals (rice, wheat, oats, and barley) and less refined products of cereals, fruits (bananas and apples), vegetables (potato, beet, carrot, bean, spinach, and lentils) (Powell et al., 2005), and beverages such as beer since the Si contained in barley and hops is solubilized during the manufacturing process (Pennington, 1991; Powell et al., 2005; Jugdaohsingh, 2007). Other sources of silicon are meat, fish, milk, and eggs (Nielsen, 1974; Nuurtamo et al., 1980). Drinking water can also be a source of Si depending on the source and the method of processing (Jugdaohsingh, 2007; Sripanyakorn et al., 2009). In plants Si is not considered an essential element, but it has been found that its inclusion in fertilizer formulations provides higher tolerance to stress (Adrees et al., 2015; Rizwan et al., 2015; Cooke and Leishman, 2016; Luyckx et al., 2017), especially on soilless growing conditions (Epstein, 1994, 2009; Voogt and Sonneveld, 2001). An additional benefit of adding silicon in the fertilization of crops is related to the more significant amount of silicon available to human consumers. In other words, the use of silicon in agricultural production brings a benefit to agricultural producers in the form of stronger and stress-tolerant plants, while for consumers of harvested products it gives an advantage in the way of higher silicon intake in the food. The use of mathematical models in the mineral nutrition of plants allows to simulate the dynamics of the absorption of water and dissolved ions in response to different internal and external factors (Juárez-Maldonado et al., 2017). The models contribute to the quantitative understanding of the factors involved in the absorption, transport, and accumulation of mineral elements; additionally, they allow to explore different environmental or endogenous situations that modify the nutrition of the plant (Mankin and Fynn, 1996). Regarding Si modeling, (Sakurai et al., 2017) presented a dynamic model of Si absorption and transport for rice. The model was integrated considering the activity of different transporters and the distribution of Si through different nodes in the entire plant; the model was able to predict the dynamic behavior of silicon in the plant successfully. However, in the case of vegetables, there are no models that consider silicon, although there are published models that effectively simulate nutrition with other mineral elements (Juárez-Maldonado et al., 2014b, 2017; Ramírez-Pérez et al., 2018). The aim of this manuscript was the integration of an estimated dynamic model that approximates the availability, absorption, transport, and accumulation of silicon in a tomato crop in soil and soilless conditions. ### BENEFITS OF SILICON IN HORTICULTURAL PLANTS The ferns, horsetails, and grasses such as corn, wheat and rice, and sugar cane, are the plants that naturally accumulate more silicon (Liang et al., 2015). However, in the presence of adequate amounts of silicon in the form of Si(OH)4, all plants, including horticultural species such as tomato and cucumber, absorb it. Plants use silicon in a manner not yet well understood to stimulate the antioxidant metabolism, the processes of plant's hardening, defense, and adaptation to environmental factors. Depending on whether they are species that carry out silicification, Si(OH)<sup>4</sup> is concentrated in polymeric form (amorphous hydrated silica) in different cellular and extracellular compartments, finally transforming it using a deposition process dependent on transpiration into insoluble biogenic silica (SiO2.nH2O) which forms structures called phytoliths or opal (Sangster et al., 2001; Katz, 2014; Exley, 2015). The biogenic silica is subsequently incorporated into the soil contributing with 1–3% of the total Si in the soil (Desplanques et al., 2006). The silicon absorbed by the plants seems to be maintained under constant exchange between the soluble forms (Si(OH)4) and the insoluble fraction (polymeric silicic acid and biogenic silica) (Exley, 2015). Most of the Si deposited as biogenic silica remains as such throughout the life of the plant (Sangster et al., 2001). The soluble part is directly available to humans when they consume plant foods, while the insoluble fraction could perhaps be considered as an integral part of the fiber. The environmental factors and differences between plant species that modify the ratio Si soluble/Si insoluble, which ultimately determines the dietary utility of the product, have not been studied. Considering that, (i) all plants seem to have the capacity to absorb silicon (Exley, 2015), (ii) and in view of the rise that has taken in recent years the production of vegetables using soilless production systems (Pignata et al., 2017), (iii) in addition to the fact that irrigation water and horticultural substrates provide little bioavailable silicon (Liang et al., 2015), then, it would be advisable to include silicon on a daily basis in fertilizer formulations used in those soils with low Si bioavailability as well as in soilless crops for the production of vegetables under protected conditions (Epstein, 1994, 2001). The different groups of plants have different capacity to mobilize Si toward their various organs, but practically all absorb the silicon from the soil when it is available in the soil solution or the nutrient solution. The species with low mobilization capacity accumulate it in the roots and stems, while the species with high mobilization capacity accumulate it in stems, leaves, fruits, and seeds. Si appears to be absorbed in the form of Si(OH)<sup>4</sup> by channels belonging to the aquaporins' group. Thus the rate of absorption and transport depends on the flow of water linked to transpiration (Exley, 2015; Sakurai et al., 2017). The cereals are plants with a high capacity of silicification and therefore represent a significant amount of Si in the diet. However, the silicon contained in cereals will be encountered almost all in the form of insoluble biogenic silica (Sangster et al., 2001) which would be partially dissolved by the acids of the digestive system; on the other hand, in fruits and vegetables, due to their lower silicification capacity, it is expected that there will be more soluble silicon, which theoretically would be more available to be assimilated during intake. Considering the above, it is possible that the fruits of horticultural species such as tomato can be excellent sources of Si for the diet. Additionally, it is known that in comparison with the dicotyledons, cereals contribute less Ca and Mg (White and Broadley, 2005). Therefore, a diet high in cereals that provides a significant amount of Si on average will contain less Ca and Mg than a mixed diet with base in cereals and dicots. On the other hand, the consumption of vegetables and fruits has grown considerably in recent decades among the human population and it is desirable that species such as tomatoes, eggplants, strawberries, cucumbers, avocados, melons, watermelons, carrots, onions, chilies, pumpkins, among others, contain a higher amount of silicon, considering the double benefit already mentioned of the crop higher tolerance to stress and the contribution of Si to human consumers. In soilless crops, it is necessary to consider the contribution of Si in fertilizers since irrigation water does not provide enough, only from 5 to 24 mg L−<sup>1</sup> Si (Liang et al., 2015). The lowest recommended concentration of Si in the nutrient solution for plants growing on substrates other than soil is 28 mg L−<sup>1</sup> Si (Epstein, 1994), which can be achieved with 123 mg L−<sup>1</sup> of Na2SiO3. ### EDAPHIC FACTORS THAT DETERMINE THE AVAILABILITY OF SILICON Si is found in soil as an inert mineral in the form of quartz or aluminosilicates such as micas and feldspars. The weathering of these materials by rainwater, irrigation water, or by the acid metabolites of microorganisms and plant roots produce Si(OH)<sup>4</sup> that under a balanced condition reaches a concentration of up to 1.8 mM (173 mg L−<sup>1</sup> , equivalent to 50.4 mg L−<sup>1</sup> Si). Above this level, reaching 2 mM (192.18 mg L−<sup>1</sup> ), Si(OH)<sup>4</sup> forms hydrated amorphous silica polymers containing Si unavailable for plants (Epstein, 2001; Liang et al., 2015). The actual value of the concentration of Si(OH)<sup>4</sup> in the soil solution is much lower than 1.8 mM, commonly found between 0.1 and 0.6 mM (9.61–57.66 mg L−<sup>1</sup> ), but with such low values as 0.02 mM (1.92 mg L−<sup>1</sup> ) in very eroded soils (Epstein, 2001; Liang et al., 2015). The concentration of bioavailable Si in soil solution results from the release rate of Si(OH)4. Bioavailability is dependent on the silicon content of the soil minerals, organic matter, the temperature, the amount of precipitation and the acidity of the soil or soil pore water. The incorporation of Si in plants occurs at a rate dependent on the intensity of the transpiration (Exley, 2015), so that conditions of rapid growth can rapidly decrease the availability of Si in the soil solution (Epstein, 2001; Liang et al., 2015). Soils of tropical areas where high precipitation occurs as well as calcareous and sandy soils of semi-arid and arid regions with low vegetation provide low quantities of Si to the soil solution, so it is recommended to use fertilizers with Si (Epstein, 1999). An affordable source of Si is siliceous sand that is offered in different granulometries, and that is used in quantities of between 500 and 4,000 kg ha−<sup>1</sup> . On the other hand, Mollisol and Vertisol soils of the temperate and subtropical regions are soils that can provide an adequate amount of silicon (Epstein, 2001; Gérard et al., 2008). However, this has not been corroborated in regards of the actual availability of Si(OH)<sup>4</sup> in soil pore water, since there is little-published information about concentrations, dynamic behavior, and association with other edaphic components of Si in the solution of the soil. The temperature exerts a substantial impact on the soil solubilization rate of Si. However, the seasonal changes in temperature are significant as a determinant of the Si concentration in the soil solution only in the cold seasons of temperate zones, because the range of temperatures suitable for the growth of a crop is also adequate for the solubilization reactions of silicon in the soil (Sommer et al., 2006). Therefore, the temperature is not considered as a factor subject to management regarding the bioavailability of Si for crop plants. Possible exceptions would be crops in soil mulching and crops grown in greenhouse soil or tunnels. In both cases, soil or substrate temperatures are more stable, and on average higher than those of uncovered soil, in addition to water management more precise in time and quantity, so the bioavailability of Si is expected to be higher. Another factor regulating the availability of Si(OH)<sup>4</sup> is the pH of the soil pore water, that depends on the pH of the rainwater or irrigation water and is also modified by the respiratory activity and extrusion of organic acids by microorganisms and plant roots (Pérez-Labrada et al., 2016). In fact, the presence of Si induces the synthesis of citric acid in plants (Hernandez-Apaolaza, 2014). The pK1 of Si(OH)<sup>4</sup> is 9.6, which indicates that its bioavailability in a nutrient solution is practically unaltered with pH values lower than 9. In the study of Gérard et al. (2008), there was little impact of pH on the bioavailability of Si in the soil solution, but the study conditions were developed under a very narrow range of pH variation. It will be necessary to collect data in different types of soil, or in soils subjected to treatments that modify its reaction or the pH of the soil pore water, to determine the effect of pH on the concentration of Si(OH)4. Both a nutrient solution and the soil solution contain components that modify pH and interact with Si. With a pH> 7 that promotes the formation of Fe hydroxides, an adsorption process occurs that causes the polymerization of Si(OH)4. With pH< 6 Si(OH)<sup>4</sup> begins to polymerize on surfaces with minerals containing Fe, while Al3<sup>+</sup> would promote the stabilization of Si(OH)<sup>4</sup> polymers, which would make Si unavailable for plants (Sommer et al., 2006). Considering this, it is possible that the availability of Si in the soil solution is higher with pH values between 6.0 and possibly 7.5 (maybe showing some resemblance to the pattern of bioavailability of P), as long as the soil parent material provides Si in sufficient quantity. Calcareous soils, which naturally have pH values> 8 in the soil solution (Pérez-Labrada et al., 2016), do not provide enough Si (Liang et al., 1994). Thus the fertilizer contributions with Si in crops in calcareous soils are beneficial (Zhang et al., 2017). Another factor to consider regarding the availability of Si(OH)<sup>4</sup> in the soil pore water is soil organic matter (SOM) and its dissolved forms. SOM have a profound impact on the availability of mineral elements (Diacono and Montemurro, 2010), either directly by chemical processes or indirectly by the promotion of bacteria and fungi that solubilize Si and other elements of soil minerals (Landeweert et al., 2001). An expected effect of SOM would be the adsorption of Al3<sup>+</sup> through organic acids (Rustad and Cronan, 1995), which would decrease the Al-Si association and increase the concentration of Si(OH)<sup>4</sup> available in the soil solution. The organic acids derived from SOM are also agents that promote dissolution in mineral surfaces (Drever and Stillings, 1997) so that in soils with silicon-rich parent materials or agricultural soils with the application of Si fertilizers would be very helpful. In nutrient solutions for soilless crops, the use of organic acids can also be useful to improve the solubility of fertilizers with silicon. On the other hand, indirect evidence is available that indicates that SOM is a factor that increases the bioavailability of Si for crop plants (Ding et al., 2008; Sun et al., 2017), thereby SOM management should be considered to increase the availability of Si for crops. ### THE MODEL The information in the previous section highlighted the factors that can be subjected to management in a crop, both in soil and soilless, with the purpose of increasing the availability of Si(OH)<sup>4</sup> for plants. In the crops grown in soil, a primary factor is the silicon content of soil's parent material. In the fertile soils of temperate and subtropical zones, Si inputs are rarely required in the fertilizers since the soil will undoubtedly provide the necessary amount. On the other hand, in the calcareous soils of arid and semi-arid regions, and in the soils of tropical regions subject to regimes of intense precipitation, the application of Si with fertilizers will be necessary, but also the consideration of the pH and organic matter management of the soil to ensure adequate availability of Si(OH)4. In soilless crops, the critical factor to consider will be the concentration of Si in the irrigation water. Values below 28 mg L −1 Si point out the need to provide Si up to a maximum of 50 mg L −1 . The management of pH is the next factor to be considered. However, the data presented indicate that pH management aimed at ensuring the bioavailability of P in nutritive solution (that is, maintaining it between 5.5 and 7) will be adequate. A Matlab-Simulink model (the archives are included in Supplementary Material) is presented below which allows verifying the impact of different environmental scenarios, both in a soil crop and in a soilless crop, using as a model tomato plants. There is also an example of the use of the software to obtain the estimated impact of the environmental variables on the absorption of Si by the tomato plants. The data presented in the previous parts of the manuscript can be tested in this model by verifying the result regarding the concentration of Si in the plants. The purposes of the use of the model are educational or technical, and from our perspective, the model can be useful in the agronomic management of Si in a tomato crop and, possibly applicable to other horticultural crops. ### Description of the Model Tomato (Solanum lycopersicum L.) was used as a biological model to describe the distribution of silicon accumulation in the different organs. To describe the effects of the various environmental factors mentioned, the deterministic mathematical model proposed by Tap (2000) and modified by Juárez-Maldonado et al. (2014b) will be used as a basis. The model consists of six state equations, using as inputs the radiation (PAR, µmol m−<sup>2</sup> s −1 ), temperature (◦C), and CO<sup>2</sup> concentration (µL L−<sup>1</sup> ). The model allows to directly considering the effect of these three variables on the accumulation of silicon in the different organs of the tomato plant. The scope of the model refers to environmental conditions where intense stress does not prevail since it is assumed that the growth rate of the plants will be a direct function of the irradiance and temperature. Within the plant, silicon accumulates in different organs depending on the corresponding transpiration rates. Thus, it is necessary to calculate the transpiration by a tomato plant dynamically. For this, the equation 1 is used, based on the fact that a linear correlation can be considered between the biomass accumulated by the tomato plant and its transpiration (Juárez-Maldonado et al., 2014a). $$Transipation = Biomass \ast plm/t\text{cg} \tag{1}$$ Biomass is the mass of the tomato plant in g m−<sup>2</sup> ; plm is a parameter of the linear model (8.5714); and tcg is the time of crop growth (10279801 s). Assuming that the tomato plant does not present a substantial accumulation of Si (Liang et al., 2015), the maximum absorption limit was set for the model at 1% (as SiO2) of the dry biomass (Miyake and Takahashi, 1978). In this condition, and while there is an unlimited availability of silicon in the soil solution, the accumulation of maximum total silicon (MSiT) in the plant would be as follows: $$\text{MSiT} = \frac{Biomass}{100} \ast PD \tag{2}$$ Where PD is the planting density expressed in plants m−<sup>2</sup> , which for this model was established in 3 plants m−<sup>2</sup> . This plant density was used by Juárez-Maldonado et al. (2014b) and provide the best financial margin, high yield, and fruit quality (Peet and Welles, 2005). The distribution of accumulated silicon in the tomato plant will then follow the different transpiration rates of its organs, that is, leaves> stem> fruits ≥ root. In the particular case of tomato, organ transpiration can be approximated to the following percentages of total transpiration: leaves = 90%, stem = 5%, fruits = 2.5%, and root = 2.5%. Even though the potential availability of Si(OH)<sup>4</sup> in the soil solution is 192.18 mg L−<sup>1</sup> (Epstein, 1999; Liang et al., 2015), disponibility is affected by temperature, pH, and organic matter content of the soil. In addition to the factors that are modified with agricultural management such as soil moisture and soil profile. According to the literature, the availability of silicon in soils is directly affected by soil temperature (Epstein, 1999; Liang et al., 2015). Although there is no clear explanation of how this behavior occurs, it is possible to approach it with a third-order model (Equation 3). This is due to the disponibility of silicon is between 8 and 35◦C, being its highest availability at 25◦ C. $$T\mathfrak{B}\Temp^3 + T\mathfrak{Z}\Temp^2 + T1\Temp + T0\tag{3}$$ Where T3, T2, T1, and T0 are the parameters of the thirdorder model (equivalent to −0.0003; 0.0127; −0.1093; and 0.1674 respectively), and Temp is the 0–30 cm soil temperature (◦C). Concerning the SOM, it is known that there is a positive correlation with the availability of silicon (Ding et al., 2008; Sun et al., 2017). View from an agricultural perspective, soil is rich in organic matter when it has a concentration of 5%. An adjustment with a Michaelis-Menten function was used to describe the higher availability of Si, due to the effect of SOM. For this, the following Equation (4) was used. $$Vma\overline{\times} \times \frac{OM}{(Km+OM)}\tag{4}$$ Where Vmax is the parameter of maximum availability of silicon due to organic matter normalized to 1 (Vmax = 1). OM is the amount of organic matter contained in the soil (%, w/w). And Km is the Michaelis-Menten parameter (Km = 2.5). The pH is also a determining factor in the availability of silicon (Liang et al., 2015). This factor, as well as temperature, is related to the availability of silicon that can be approached to a thirdorder model. The availability of silicon in soil occurs in the pH range from 2 to 9, with a pH of 7 being the highest availability. Therefore, its effect can be described as follows: $$pH\text{3} \* pH^{3} + pH\text{2} \* pH^{2} + pH\text{1} \* pH + pH\text{0}\tag{5}$$ Where pH3, pH2, pH1, and pH0 are the parameters of the thirdorder model (with values −0.0235, 0.325,−1.1563, and 1.2262, respectively). And pH represents the pH of the soil studied. Therefore, the Si(OH)<sup>4</sup> available (SiAv) to be absorbed by the tomato plant is described by the following equation: $$\text{SiAv} = (\text{SiP} - \text{SiWater}) \ast ETem \ast EOM \ast EpH + \text{SiWater} \quad \text{(6)}$$ Where SiP is the maximum amount of silicon in a soil without polymerization [192.18 mg L−<sup>1</sup> Si(OH)4]; ETem represents the effect of temperature on the availability of silicon (Equation 3); EOM describes the impact of organic matter on the availability of silicon (Equation 4); EpH represents the effect of pH on the availability of silicon (Equation 5); and SiWater is the amount of Si(OH)<sup>4</sup> available in the irrigation water. The model supposes that under no condition will be the available Si(OH)<sup>4</sup> be higher than the SiP value. The accumulated Si (as SiO2) in the tomato plant (SiT) was determined with the silicon [Si(OH)4] available and the transpiration (Equation 1) in the following way: $$\text{SiT} = \text{SiAv} \ast \text{Transjunction} \ast \text{SiSi} \tag{7}$$ Where SiSi is the fraction of cumulative Si in the plant of the total available Si(OH)<sup>4</sup> (g). In soilless cultivation conditions, the only source of Si(OH)<sup>4</sup> for the crop will be the content of the irrigation water since there is no such source of soil mineral replacement as in the soil. Therefore, the accumulation of Si in the tomato plant in soilless culture (ASiTSC) will depend entirely on the transpiration of the plant (Equation 1) and the availability of Si(OH)<sup>4</sup> in the irrigation water (SiWater). This relationship is expressed as: $$\text{ASiTSC} = \text{Transjunction} \ast \text{SiWater} \ast \text{SiSi} \tag{8}$$ As previously described, the availability of silicon in soil depends on three primary conditions: pH, organic matter, and soil temperature. Of these conditions, it is feasible to modify the amount of organic matter or the pH. In the case of temperature, the easiest way would be to use covers as plastic mulches, which could increase the soil temperature by 3–4◦C (Ruíz-Machuca et al., 2015). Therefore, these factors can be considered as crucial factors to the agronomic management of silicon availability (Liang et al., 2015). ### Silicon Accumulation in Tomato According to the simulations carried out using the proposed model, a soil with pH 7.0 and organic matter content of 6% can obtain the maximum availability of Si(OH)4, which can be > 4,500 mg m−<sup>2</sup> at 15◦C; or > 8,300 mg m−<sup>2</sup> at 25◦C (Figures 1A,D). Considering the availability, and two average temperature conditions of soil (15 and 25◦C), the highest availability of silicon is obtained with an average soil temperature of 25◦C (Figure 1D). On the contrary, when the organic matter content is low (<1%) along with a pH >8 (like a soil of a semi-arid region), the availability of Si(OH)<sup>4</sup> in the soil can drastically decrease to <20 mg m−<sup>2</sup> at 15◦C (Figure 1A), or <34 mg m−<sup>2</sup> at 25◦ C (Figure 1D). These results describe the effect of pH, organic matter, and temperature factors on the availability of Si(OH)<sup>4</sup> in soil. In addition to demonstrating the potential sensitivity of the availability of SiOH<sup>4</sup> in the soil to the modifications on any of the conditions mentioned. The availability of silicon in the soil will directly impact the accumulation of plants grown on it (Epstein, 2001). The higher availability of silicon derived from the factors evaluated (Figure 1D), results in a more significant accumulation of Si (as SiO2) based on the dry weight of the tomato plant (up to 1 %), as can be seen in Figure 1E. The Figure 1E fits well the reported Si concentration in tomato plants grown under pH 5.5 (Miyake and Takahashi, 1978) to pH 8.48 (Gunes et al., 2007). The same behavior is observed concerning accumulated silicon per plant, reaching up to 13.7 g per plant, that represent the maximum accumulation of silicon for tomato plants under this conditions (Figure 1F). However, low availability of silicon in the soil can lead to a small accumulation of silicon in the plant. In the example the conditions of a soil corresponding to the situation of a semi-arid region, there would be an accumulation <1.3 g per plant for both soil temperature conditions (Figures 1C,F). This equals to a silicon concentration based on plant dry weight <0.1% (Figures 1B,E). Since silicon accumulates in the different organs of the tomato plant as a function of the rate of transpiration, then the highest accumulation will be observed in the leaves, since they represent 90% of total transpiration. In fruits, a lower accumulation of silicon will be seen, since the rate of transpiration is little compared to that of the leaves (∼5%) (Leonardi et al., 2000). However, the availability of silicon in the soil will ultimately define the accumulation of silicon both in the entire plant and in its various organs. Contrary to a crop established in soil, in soilless cultivation, e.g., hydroponics, the primary factor that will modify the availability of Si for the plant will be its concentration in the irrigation water used. It has been reported that irrigation water can have a Si content [as Si(OH)4] of 5–20 mg L−<sup>1</sup> , while it is considered that an adequate concentration of Si would be 28 mg L −1 (Epstein, 1994, 1999; Liang et al., 2015). However, the transpiration rate of the crop will finally define the amount of SiOH<sup>4</sup> absorbed and accumulated in the different organs. Since the growth of the plant and the proper distribution of biomass in the various organs will affect the rate of transpiration, then crop growth should be considered as an additional factor that will change the accumulation of silicon in a soilless crop system. Therefore, the environmental factors (PAR, CO2, air temperature) that affect the growth of the crop will, in turn, affect the accumulation of silicon in the different organs. According to the simulations carried out, a low concentration of CO<sup>2</sup> and a low incidence of PAR generate little accumulation of biomass in the tomato plant (Figures 2A,D). This same result is observed when the temperature of the environment changes, 30◦C generates biomass of up to 3,000 g per plant (Figure 2A); while 20◦C produces up to 2,240 g per plant (Figure 2D), at the highest conditions of PAR and CO<sup>2</sup> concentration. As a consequence, the total transpiration of the plant is modified when environmental factors changes, and therefore the accumulation of silicon. With an average temperature of 30◦C, the biomass accumulated in the fruits can represent around 60% of the total of the tomato plant, and the leaves less than 10%. However, when the temperature drops to 20◦C, the biomass distribution in the tomato plant changes. Under this condition, the biomass accumulated in the fruits is ∼33%, whereas in the leaves it increases up to 33%. Since the leaves constitute the largest area of transpiration, modifying the temperature of the environment in which the tomato grows substantially alters the final accumulation of silicon. Therefore, according to the model, a temperature of 30◦C will lead to a lower accumulation of silicon in the tomato plant (Figure 2B), while at 20◦C there will be more significant accumulation (Figure 2E). The Figure 2C fits the reported Si concentration in tomato plants grown under temperatures of 28–32◦C (Cao et al., 2015). The result will be a higher level of silicon in dry weight of the tomato plant at low temperatures (Figure 2F), while high temperatures will decrease the concentration considerably (Figure 2C). When considering the availability of silicon in the irrigation water, it can be observed that a condition of low Si content (5 mg L−<sup>1</sup> as Si[OH]4) will result in less concentration and accumulation of silicon in the plant (Figures 3A,B). On the contrary, adequate availability of Si (28 mg L−<sup>1</sup> as Si[OH]4) in the irrigation water will result in increased accumulation and concentration of silicon in the plants (Figures 3C,D). High accumulation appears to occur regardless of the environmental conditions in which the crop develops, when there is adequate availability of Si in the irrigation water. However, when conditions are favorable for the development of leaves in tomato plants, the maximum concentration of silicon for this species can be reached (Figure 3D). The model presented focuses on the impact of external factors on the growth of tomato plants, under the assumption that as long as exists the availability of Si the plants absorb it and transport it at a rate proportional to the growth rate and transpiration rate. The point highlighted with the model is that the biofortification of the fruits with Si depends on the availability of the element in the form of Si(OH)<sup>4</sup> both in soil and in soilless culture. Sakurai et al. (2017) developed a successful dynamic model of Si absorption and transport for rice. The model is based on endogenous variables, as the activity of different transporters and the distribution of Si through different nodes in the entire plant. The model presented in this manuscript is focused on exogenous variables, susceptible to agronomic management both in field cultivation as in the greenhouse, and it has been used successfully (C,D). For the simulation, 12 h of PAR and an average air temperature of 20◦C were considered. to simulate the absorption of other elements in tomato and other crops (Juárez-Maldonado et al., 2014b; Ramírez-Pérez et al., 2018). However, it must be taken into account that the presented model is used to describe the accumulation of silicon in the plant under relatively favorable environmental situations. The presence of stresses such as water deficit, salinity, deficiency of mineral nutrients and pathogens, results in loss of precision. With a certain amount of PAR and with a particular temperature regime, the stressed plants would have real biomass lower than the estimated by the model, which means an overestimation of the absorbed silicon. As far as we know, except those published by (Sakurai et al., 2015, 2017) for monocotyledons, there are no similar dynamic models published about the absorption of silicon in dicots. The model described for the tomato crop is a first preliminar advance that we believe substantially can improve the understanding of some factors that regulate the bioavailability of silicon. ### CONCLUSIONS After the results obtained from the presented model, the following is proposed: 1) When crops are grown in soil, the bioavailability of silicon can be increased by adding organic matter from organic amendments or humic substances, or by modifying the pH of the soil solution, using organic or inorganic acids, to be the closest to 7.0. ### REFERENCES ## AUTHOR CONTRIBUTIONS All authors were responsible for processing information and manuscript writing. AB-M, AJ-M, FP-L, and AM-D: Conceptualization; AJ-M, ML-P, LR-P, and AM-D: Model design and implementation; ML-P, FP-L, SG-M, LG-D, JG-M, and AB-M: Manuscript drafting. All authors read and approved the final manuscript. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00647/full#supplementary-material Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 López-Pérez, Pérez-Labrada, Ramírez-Pérez, Juárez-Maldonado, Morales-Díaz, González-Morales, García-Dávila, García-Mata and Benavides-Mendoza. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Genetic Basis of Variation in Rice Seed Storage Protein (Albumin, Globulin, Prolamin, and Glutelin) Content Revealed by Genome-Wide Association Analysis Pingli Chen<sup>1</sup> , Zhikang Shen<sup>1</sup> , Luchang Ming<sup>1</sup> , Yibo Li<sup>1</sup> , Wenhan Dan<sup>1</sup> , Guangming Lou<sup>1</sup> , Bo Peng<sup>1</sup> , Bian Wu<sup>1</sup> , Yanhua Li<sup>2</sup> , Da Zhao<sup>1</sup> , Guanjun Gao<sup>1</sup> , Qinglu Zhang<sup>1</sup> , Jinghua Xiao<sup>1</sup> , Xianghua Li<sup>1</sup> , Gongwei Wang<sup>1</sup> and Yuqing He<sup>1</sup> \* <sup>1</sup> National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, China, <sup>2</sup> Life Science and Technology Center, China National Seed Group Co., Ltd., Wuhan, China ### Edited by: Huixia Shou, Zhejiang University, China ### Reviewed by: Umesh K. Reddy, West Virginia State University, United States Shahidul Islam, Murdoch University, Australia > \Correspondence: Yuqing He [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 21 January 2018 Accepted: 18 April 2018 Published: 09 May 2018 #### Citation: Chen P, Shen Z, Ming L, Li Y, Dan W, Lou G, Peng B, Wu B, Li Y, Zhao D, Gao G, Zhang Q, Xiao J, Li X, Wang G and He Y (2018) Genetic Basis of Variation in Rice Seed Storage Protein (Albumin, Globulin, Prolamin, and Glutelin) Content Revealed by Genome-Wide Association Analysis. Front. Plant Sci. 9:612. doi: 10.3389/fpls.2018.00612 Rice seed storage protein (SSP) is an important source of nutrition and energy. Understanding the genetic basis of SSP content and mining favorable alleles that control it will be helpful for breeding new improved cultivars. An association analysis for SSP content was performed to identify underlying genes using 527 diverse Oryza sativa accessions grown in two environments. We identified more than 107 associations for five different traits, including the contents of albumin (Alb), globulin (Glo), prolamin (Pro), glutelin (Glu), and total SSP (Total). A total of 28 associations were located at previously reported QTLs or intervals. A lead SNP sf0709447538, associated for Glu content in the indica subpopulation in 2015, was further validated in near isogenic lines NIL(Zhenshan97) and NIL(Delong208), and the Glu phenotype had significantly difference between two NILs. The association region could be target for map-based cloning of the candidate genes. There were 13 associations in regions close to grainquality-related genes; five lead single nucleotide polymorphisms (SNPs) were located less than 20 kb upstream from grain-quality-related genes (PG5a, Wx, AGPS2a, RP6, and, RM1). Several starch-metabolism-related genes (AGPS2a, OsACS6, PUL, GBSSII, and ISA2) were also associated with SSP content. We identified favorable alleles of functional candidate genes, such as RP6, RM1, Wx, and other four candidate genes by haplotype analysis and expression pattern. Genotypes of RP6 and RM1 with higher Pro were not identified in japonica and exhibited much higher expression levels in indica group. The lead SNP sf0601764762, repeatedly detected for Alb content in 2 years in the whole association population, was located in the Wx locus that controls the synthesis of amylose. And Alb content was significantly and negatively correlated with amylose content and the level of 2.3 kb Wx pre-mRNA examined in this study. The associations or candidate genes identified would provide new insights into the genetic basis of SSP content that will help in developing rice cultivars with improved grain nutritional quality through marker-assisted breeding. Keywords: GWAS, storage protein, grain quality, endosperm, nutrition, Oryza sativa L. ### INTRODUCTION fpls-09-00612 May 7, 2018 Time: 23:5 # 2 Rice is one of the major staple cereal foods and is an important source of total protein in human food. SSP account for approximately 8% of the dry grain weight and are the second most abundant ingredient after starch in rice. Rice has the lowest protein content among cereal grains, but net protein utilization is highest (Juliano, 1992). High-protein rice is likely to increase human nutrition in poor families, especially where rice is a staple food. Therefore, increasing the SSP content has become one of the main breeding objectives in improving nutritional quality in rice. The SSP in rice can be classified into four fractions: albumin, globulin, prolamin, and glutelin, according to differences in solubility. Glutelin encoded by 15 genes accounts for as much as 80% of the total SSPs and is concentrated in the milled fraction, whereas prolamin, the most evenly distributed protein, accounts for less than 5% (Yamagata et al., 1982). Based on amino acid sequence similarity, glutelins are classified into four subfamilies: GluA, GluB, GluC, and GluD (Kawakatsu et al., 2008). In rice, SSP genes have been cloned and characterized mostly by mutant screening (Ren et al., 2014). Glutelins are synthesized in the rough endoplasmic reticulum as a 57 kDa precursor. Previous studies have identified rice 57H mutants that accumulate relatively high levels of 57 kDa pro-glutelin and have floury/opaque endosperm phenotype (Wang et al., 2009; Ren et al., 2014). Of 57H mutants, only gpa3, Osvpe1, and OsRab5a have been successfully cloned (Wang et al., 2009; Wang Y. et al., 2010; Ren et al., 2014). Prolamins are encoded by a multigene family of 34 gene copies. Based on the molecular mass, prolamins classified into three groups: 10 kDa prolamin (RP10), 13 kDa prolamin (RM1, RM2, RM4, and RM9), and 16 kDa prolamin (RP16) (Yamagata et al., 1982; Kawakatsu et al., 2008). Both albumin and globulin are concentrated in the bran and polishing during milling removes a major portion of these proteins (Shewry, 2007). Globulin is also easily digested (Yamagata et al., 1982; Zhang et al., 2008), and only a limited number of genes has been cloned and characterized (Bhullar and Gruissem, 2013). RA16 and RA17 that are associated with seed allergenic protein have been reported as albumin genes in previous studies (Adachi et al., 1993). The nutritional value of glutelin is higher than prolamin because it has a greater digestive capacity by humans and higher lysine content in rice (Ogawa et al., 1987). However, patients with kidney disease and diabetes need low glutelin diet (Mochizuki and Hara, 2000; Nishimura et al., 2005; Morita et al., 2009). Some proteins that belong to the albumin and globulin are considered to be allergenic. Collectively, emphasis in rice breeding should not only be on the concentration, but also on the quality of rice protein. Seed storage protein is quantitatively inherited, and is affected by growing environment (Shewry, 2007). QTL mapping based on molecular markers and linkage maps has always been a common method of genetic studies (Li et al., 2014; Peng et al., 2014). Many QTL for crude protein content in rice have been reported (Aluko et al., 2004; Wang et al., 2008; Lou et al., 2009; Yu et al., 2009; Peng et al., 2014), but fewer studies have investigated the individual protein fractions in milled rice. qPC1 (OsAAP6), controlling the natural variation in SSP content, has been cloned using a mapbased cloning strategy (Peng et al., 2014). Zhang et al. (2008) identified 16 QTL for contents of crude protein and the four protein fractions. Genome-wide association study (GWAS) by means of single nucleotide polymorphism (SNP) has become the method of choice for investigation of the genetics of important traits in Arabidopsis thaliana (Chan et al., 2011), rice (Huang et al., 2010), maize (Xiao et al., 2016), sorghum (Morris et al., 2013), and others (Ogura and Busch, 2015). Although GWAS is widely used in genetic analysis of grain quality traits in rice, such as gelatinization temperature, amylose content, grain appearance, and milling quality (Borba et al., 2010; Huang et al., 2010), few studies have used this approach to investigate total SSP in rice (Huang et al., 2012; Bryant et al., 2013). The analysis of genetic basis of nutritional quality has been reported in maize (Deng et al., 2017). Maize opaque2 (o2) mutation could increase free lysine levels and create the foundation for quality protein maize (QPM) breeding. Combining GWAS and linkage mapping, a gene duplication at the 27-kDa γ-zein locus qγ27 was identified (Liu et al., 2016). qγ27 increases the level of 27-kDa γ-zein gene expression in QPM, which is essential for endosperm modification. GWAS on amino acids has been carried, and 247 and 281 significant loci were identified in two different environments (Deng et al., 2017). However, no studies have been reported about the genetic basis of the four SSP fractions by GWAS in rice. In this study, we performed GWAS of Alb, Glo, Pro, Glu, and total SSP in milled rice using 527 Oryza sativa accessions grown in two environments with an aim to identify loci involved in the genetics. Haplotype analysis and expression pattern of candidate genes then provided valuable in better understanding the genetic basis of variation in SSP content. The results shown here promote our understanding of the genetic basis of the storage protein groups, should be of use for breeders attempting to improve nutritional quality by means of marker assisted selection. ### MATERIALS AND METHODS ### Plant Materials, Field Experiments, and Trait Measurements A diverse worldwide collection of 527 O. sativa landraces and elite accessions (Supplementary Table S1) was used in this study. Structural analysis indicated that the entire collection belonged to nine subpopulations: indI, indII, indica intermediate, Tej, Trj, japonica intermediate, Aus, VI, and intermediate (Chen et al., 2014) and are available at the RiceVarMap<sup>1</sup> . The indica subpopulation (indI, indII, and indica intermediate) included 294 accessions, whereas the japonica subpopulation (Tej, Trj, and japonica intermediate) included 155 accessions. Lines were planted in two environments in Hubei province: Ezhou in 2014 (Env. 1) and Wuhan in 2015 (Env. 2). The sowing dates were 25 May in both years. Seedlings about 25 days old were transplanted to the field. There were three <sup>1</sup>http://ricevarmap.ncpgr.cn rows with 10 plants each in each plot. The planting density was 16.5 cm between plants within a row, and 26.4 cm between rows. Field management basically followed recommended practice of agriculture, with fertilizer applied (per hectare) as follows: 48.75 kg nitrogen, 58.5 kg phosphorous, and 93.75 kg potassium as the basal fertilizer; 86.25 kg nitrogen at the tilling stage; and 27.6 kg nitrogen at the booting stage. At maturity, three plants in the middle of the second row of each accession were harvested and bulked. Dry seeds were threshed in bulk and the rough rice was air-dried, and stored at room temperature for 3 months, and then stored at 4◦C. 50 g of rough rice were dehulled into brown rice using a TR 200 dehuller (Kett, Tokyo, Japan). The embryo and aleurone layer of brown rice were removed into milled rice through a Pearlest mill (Kett, Tokyo, Japan). The rice was ground into flour with a CT 410 Cyclotec mill (FOSS, Hillerod, Denmark), passed through an 80-mesh sieve and stored at −20◦C until the Alb, Glo, Pro, and Glu contents were determined based on previously published previous methods (Kumamaru et al., 1988). Briefly, 0.1 g sample of milled rice flour was placed in a centrifugation tube with 1.0 ml solvent containing 10 mM Tris–HCl buffer (pH7.5) for Alb extraction; 1.0 ml solvent containing 1 M NaCl, for Glo extraction; 60% n-propanol containing 1 mM EDTA-2Na, for Pro extraction; and 0.05 M NaOH for Glu extraction. The mixture was stirred for 2 h at room temperature, and extracts were separated from residues by centrifugation at 12,000 rpm for 15 min at 4◦C The procedure was repeated three times. The extracts were stored at −20◦C until further analysis. The contents of each fraction were determined by the Coomassie brilliant blue G-250 dye-binding method (Bradford, 1976) using bovine serum albumin as a standard, and quantitative analysis was carried out using Infinite M200 (Tecan Group, Männedorf, Switzerland) (Peng et al., 2014). Total SSP was the sum of the Alb, Glo, Pro, and Glu contents. The 2-year field experiment was designed with three replicates per year. The average SSP contents across three replicates within 1 year were used for GWAS. The SSP contents of the 527 O. sativa accessions are listed in Supplementary Table S1. Amylose content was measured as previously described method (Tan et al., 1999). ### Genome-Wide Association Study All 527 accessions were genotyped via sequencing (Chen et al., 2014). SNP information was available on RiceVarMap (see foot note text 1), a comprehensive database for rice genomics. The physical locations of the SNPs were identified based on the rice annotation version 6.1 of variety Nipponbare from Michigan State University. A total of 3,916,415 SNPs in the whole population; 2,767,159 SNPs in the indica subpopulation; and 1,857,845 SNPs in the japonica subpopulation (minor allele frequency ≥0.05; number of accessions with minor alleles ≥6) was used for GWAS (Chen et al., 2014). A linear mixed model (LMM) was used for detecting associations using Fast-LMM (Lippert et al., 2011). Population structure was controlled using a kinship matrix constructed with all SNPs (Chen et al., 2014). Effective independent SNPs were detected (Li et al., 2012), and were 757,578 in the whole population; 571,843 in the indica subpopulation; and 245,348 in the japonica subpopulation. The thresholds were set at a P-value of 5.0 × 10−<sup>6</sup> to identify significant association signals. To obtain independent association signals, multiple SNPs, exceeding the threshold in a 5 Mb region, were clustered based on an r <sup>2</sup> of LD ≥ 0.25, and SNPs with the minimum P-value in a cluster were deemed to be lead SNPs. ### Statistical Analysis Based on the standardized disequilibrium coefficients (D'), linkage disequilibrium (LD) was investigated. LD heatmaps were constructed using the TASSEL5.0<sup>2</sup> program and R package "LDheatmap"<sup>3</sup> . Statistical analysis, including a correlation analysis, was conducted using IBM SPSS Statistics 22.0. Differences in SSP values were examined by Student's t-tests. Broad-sense heritability (H<sup>2</sup> ) for each phenotype was estimated using repeatability between 2 years of phenotypic data, calculated as the variance among variety grand means divided by their total phenotypic variance. ### Candidate Genes and Haplotype Analysis Candidate genes within a 200 kb genomic region ( ± 100 kb from the lead SNP) in the associated loci were selected based on (i) biochemically related proteins or protein clusters; (ii) homologous genes with known function, and (iii) expression profiles. The genotypes of RP6, RM1, Wx, PROLM1, and other three candidate genes in the 527 rice accessions were obtained from the RiceVarMap database (see foot note text 1). The haplotypes were classified according to all SNPs (except sites in intron) including their intragenic region and 2 kb upstream with an MAF > 0.05 in a candidate gene. There were at least five rice accessions in the haplotypes for comparative analysis. One-way ANOVA and Student'st-tests were applied to compare differences in SSP content among all possible haplotype pairs. ### RNA Extraction and Quantitative RT-PCR Analysis According to the manufacturer's instructions, the total RNA was extracted from rice different tissues using TRIzol reagent (Invitrogen). About 3 µg of RNA sample was processed by RNase-free DNaseI (Invitrogen) and reverse transcribed using M-MLV reverse transcriptase (Invitrogen) with Oligo(dT)15. Quantitative RT-PCR was carried out using Fast Start Universal SYBR Green Master (Rox) superMIX (Roche, Mannheim, Germany) in a ViiA 7 Real-Time PCR system (Applied Biosystems), according to the manufacturer's introductions. Measurements were obtained using the relative quantification method. Actin was used as a reference gene in the qRT-PCR experiments. The experiment was designed with three biological replicates and three technical replicates per material. Error bars indicate standard error. The measurements were obtained using the relative quantification method. The significant difference was analyzed statistically by One-way ANOVA and Student's t-tests. All primers for qRT-PCR analysis are listed in Supplementary Table S2. <sup>2</sup>https://tassel.bitbucket.io/ <sup>3</sup>https://www.r-project.org/ ### RESULTS fpls-09-00612 May 7, 2018 Time: 23:5 # 4 ### Phenotypic Variation and Heritability of SSP Content The result analysis revealed a large variation in all phenotypes evaluated and the traits appeared to be normally distributed (Figures 1A–E* and Supplementary Table S3). Average SSP contents in Env. 1 and 2 were 70.9 and 53.7 mg/g; Alb was 2.9 and 3.3 mg/g; Glo was 5.8 and 5.3 mg/g; Pro was 2.8 and 2.1 mg/g; and Glu was 59.4 and 43.5 mg/g, respectively (Figures 1A–E). Glu accounted for approximately 80% of total SSP; Alb and Pro each accounted for about 5%; and Glo accounted for about 10% (Figure 1F). Compared with other three storage protein contents, the average content of Pro was the lowest, but the variation of Pro was largest in the whole population and each subpopulation in both environments. In four storage protein, Total SSP showed the lowest heritability (29.5%), whereas Pro showed the highest (76.8%) (Supplementary Table S3). Correlation coefficients between each pair of components, and between components and total SSP were significant and positive in both environments, except for those between Glo and Pro in any environment and between Alb and Glo and between between Alb and Pro in Env. 2 (Supplementary Table S4). High correlations were found only between Glu and total SSP in both environments with coefficients of 0.99 in Env. 1 and 0.98 in Env. 2. ### Genome-Wide Association Study for SSP Contents We performed GWAS on the entire population and on the indica and japonica subpopulations for each year. The FaST-LMM program reduced the effect of population structure (Yang et al., 2014). Quantile-quantile plots of all five traits for the whole population, and indica and japonica subpopulations are illustrated in Figure 2 and Supplementary Figures S1, S2. Some associations were detected in different subpopulations, and some of the associations for different traits were in the same chromosomal regions. Any two lead SNPs within a 100 kb region were considered to be a single association locus. The association analysis for the whole population identified 34 loci (phenotypic variance >10%) associated with three traits with a suggestive threshold value at 5.0E-06 (Table 1). Most of them were detected for Alb (21 associations) and Pro (12 associations). Lead SNPs for Alb were widely distributed in the rice genome: chromosome 1, 2, 3, 4, 5, 6, 7, 8, and 9, with chromosomes 6 and 7 having more associations. Associations explained phenotype variation of 10.4–20.9%, with the association on chromosome 6 (sf0601764762) making the largest effect. For Pro, associations accounting for 10.1–34.6% of the phenotypic variance were identified on chromosomes 2, 5, 7, 10, and 11, with chromosome 5 and 7 exhibiting more associations. Only one lead SNP sf0317000156 on chromosome 3 with a phenotype variation of 14.4% was detected for Glo. No lead SNP with phenotype variation of more than 10% was detected for Glu or total SSP. A large number of peaks (phenotypic variance >10%) were also detected by GWAS in the indica (33 associations) and japonica (40 associations) subpopulations (Supplementary Table S5). For Alb, 23 associations were distributed on all 12 rice chromosomes, except chromosome 3 and 10, but only three associations were identified in the indica subpopulation. Alb associations identified in the indica and japonica subpopulations explained phenotype variation of 10.5– 12.3, and 11.3–41.6%, respectively. Four SNPs, sf0142207782, sf0209990680, sf0605251091, and sf0804866973 individually explaining more than 30% of the Alb variation, were detected in the japonica subpopulation in Env. 1. For Glo, 14 associations were identified on chromosomes 1, 3, 4, 5, 6, 9, 11, and 12, and equal numbers of associations were detected in the indica and japonica subpopulations, explaining 10.3–16.8 and 12.9–17.0% of the variation, respectively. For Pro, there were 23 associations, involving chromosomes 1, 3, 4, 5, 7, 9, 11, and 12. Among them 14 (with phenotype variation of 10.4–38.3%) and 9 (with phenotype variation of 15.5–22.6%) associations were identified in the indica and japonica subpopulations, respectively. Ten associations for Glu were detected on chromosomes 1, 4, 5, 6, 7, 8, 9, and 10, in both subpopulations. For total SSP, only three associations on chromosomes 10 and 11 with phenotype variation of 10.4–14.8% were detected in the indica subpopulation. Among the 107 associations detected in the whole population and in the indica and japonica subpopulations, 16 were detected in different populations and nine involved two or three different traits (Table 1 and Supplementary Table S5). Examples include lead SNP sf0519612378 with phenotypic variance of 14.9– 36.6% that was detected in the whole population and indica subpopulation in Env. 1 and 2; and lead SNP sf1022972496 detected in indica subpopulation in Env. 1 was associated with phenotypic variances of 12.0 and 14.8% for traits Glu and total SSP, respectively. Seven associations in the whole population and four associations in the indica subpopulation were detected both in both environments. For Pro, two lead SNPs (sf0514987630 and sf0515211855) in the whole population and one lead SNP (sf0515706446) in the indica subpopulation were detected in both environments. The significance levels of the associations ranged from P = 5.0E-06 to P = 8.6E-16, P = 4.9E-06 to P = 9.0E-08, P = 5.0E-06 to P = 1.3E-17, P = 2.1E-06 to P = 1.4E-08, and P = 4.5E-06 to P = 3.1E-08 in LMM for Alb, Glo, Pro, Glu, and total SSP, respectively, and the most significant association for sf0706363663 located in chromosome 7 (Table 1). ### Co-localization of Associated Sites With QTLs Previously Reported and Grain Quality-Related Genes There were many overlaps between the present associations detected by GWAS and reported QTLs or intervals related to SSP content in rice. A total of 28 associations from this study were located at previously reported QTLs or intervals (shown in Table 1 and Supplementary Table S5 with corresponding references) of which 10 and eight associations were for Alb and Pro, respectively. In the indica subpopulation, lead SNP sf0709447538 in a notable hotspot region at the interval of 9.1–9.5 Mb, explaining 12.7% of the Glu variation, was detected on chromosomes 7 in 2015 (Figures 3A,B and Supplementary Table S5). Interestingly, we found that the lead SNP sf0709447538 was overlapped with the amino acid content QTLs (7–4, 7–5, and 7–6), identified in a previous study using an F<sup>9</sup> recombinant inbred line population, which derived from a cross between Zhenshan97 (ZS97) and Delong208 (DL208) (Wang et al., 2008; Zhong et al., 2011). To validate the QTL, we developed near-isogenic lines (NILs) (Figure 3C). NILs of QTL were developed by successive crossing and backcrossing ZS97 (high protein content) and DL208 (low protein content), three times (BC3) to ZS97. The QTL was selected by two molecular markers MRG186 and MRG4499 (Supplementary Table S2). Self-pollinating the BC3F<sup>1</sup> plants heterozygous for this fragment produced NIL(ZS97) and NIL(DL208). Analysis of NIL(ZS97) and NIL(DL208) showed that NIL(ZS97) was significantly higher Glu than NIL(DL208), which was the same as the phenotype in two parents (Figure 3D). The result indicated that the QTL was reliable, which helps further identify the underlying genes and their genetic basis. On the other hand, 13 associations were detected in regions close to previously identified grain-quality-related genes. Five genes (PG5a, Wx, RM1, RP6, and AGPS2a) were less than 20 kb from lead SNPs. PG5a, RM1, and RP6 were associated for Pro; Wx (P-value). Lead SNPs in significant peaks are red. The horizontal dotted line indicated the genome-wide significance threshold (P = 5.0E-06). Total, total SSP content. and AGPS2a was associated with Alb. Glutelin genes GluA1 and OsAAT2 were associated with total SSP in Env. 2. Additionally, several lead SNPs were located close to starch-metabolism-related genes, such as PUL, ISA2, Wx, GBSSII, OsACS6, and AGPS2a. SNPs located close to other reported grain quality-related genes (Prol14, RA17, RA16, and PGL) were also examined using a LMM (Table 1 and Supplementary Table S5). ### Haplotype Analyses for the Reported Genes RP6 and RM1 The association SNP sf0705739605 for Pro was each <10 kb away from two prolamin genes RP6 and RM1 reported to encode prolamins in rice (Wen et al., 1993; Kawakatsu et al., 2009). In particular, the lead SNP with highly significant P-values (P = 6.5E-11 in Env. 1 and P = 5.4E-16 in Env. 2) was identified in the whole population and indica subpopulation (Figures 4A–C, Table 1, and Supplementary Table S5). The association explained 24.7 and 26.3% of the phenotypic variances in the whole population in Env. 1 and 2, respectively. Lead SNPs sf0705735351 and sf0705739605 and all polymorphic sites in RP6 and RM1 were in high linkage disequilibrium (in high LD with each other; r <sup>2</sup> = 0.94–0.99) with most polymorphic sites (Figure 4H). We performed haplotype analyses for RP6 and RM1 and identified three main haplotypes (Hap1-3) at each locus (Supplementary Table S6). Hap1 and hap2 of RP6 and hap2 and hap3 of RM1 were not identified in japonica. Hap2 of RP6 and hap3 of RM1 had significantly higher Pro than the alternative haplotypes in both TABLE 1 \| Associated single nucleotide polymorphisms (SNPs) identified by linear mixed model (LMM) method in the whole population. Pop, population; Chr., chromosome; P, P-value estimated in LMM; P.V (%), proportion of phenotypic variance explained; Total, total seed storage protein (SSP) content. <sup>a</sup>Associated SNPs within 100 kb for the same trait are considered the same locus; for Alb, Glo, and Pro, only lead SNPs with P.V (%) more than 10% are shown; bold scripts indicate detections in different populations and italic indicates detection in different traits.bNegative value means the gene is upstream of the SNP site.cRefers to interval or QTL reference. environments (Figures 4D,F). In the region contained coding region and 2 kb upstream of RP6 and RM1, 17 and 26 SNPs were found, respectively (Supplementary Table S6). At the RP6 locus, there were two synonymous SNPs, two non-synonymous SNPs in the exon, three SNPs in the 3<sup>0</sup> untranslated regions, and 10 substitutions in the 2 kb cis-regulatory region. At the RM1 locus, there were five synonymous SNP, no non-synonymous SNPs in the exon, two SNPs in the 5<sup>0</sup> and 3<sup>0</sup> untranslated regions, and 19 plots of LMM for Pro in the all accessions in 2015 (A). (B) Local Manhattan plot surrounding the peak in 2015 on chromosome 7. Arrow indicates the position of the lead peak. The corresponding colors of r 2 represent linkage disequilibrium levels. (C) Plant architectures of near-isogenic lines. (D) Phenotypes of Glu of two parents ZS97, DL208, NIL(ZS97), and NIL(DL208). ∗∗Indicates the differences of Glu between two materials are significant at P < 0.01. substitutions in the 2 kb cis-regulatory region. To get an overview of the expression profiles of RP6 and RM1, the CREP database<sup>4</sup> , a website that contains the dynamic gene expression profile of indica rice, was searched (Wang L. et al., 2010). The results showed that RP6 and RM1 displayed high-level expressions in endosperm but low-level expressions in other tissues in ZS97 (Figures 4E,G). Considering the complexity of population structure and genetic background, we checked the expressions of RP6 and RM1 with different haplotypes in 20 and 34 accessions randomly chosen from indica group, respectively. Using qRT-PCR analysis, The results indicated that in the endosperm at 7 days after pollination (d.a.p.), expression levels of RP6 in hap2 accessions were much higher than those in hap3 accessions (Figure 4I and Supplementary Table S7), and expression levels of RM1 in hap3 accessions were much higher than those in hap1 accessions (P < 0.01) (Figure 4J and Supplementary Table S7). These results show that two genes might be good candidates for the GWAS locus. In conclusion, two genes had high-level and specific expressions in endosperm of rice, and genotypes with ### Analysis of One Candidate Gene Wx We also found a highly significant association signal for Alb involving sf0601764762 (P = 2.0E-08 in Env. 1 and P = 8.6E-16 in Env. 2) on chromosome 6 (Table 1 and Figures 5A–C). The lead SNP, explaining 12.9 and 20.9% of the phenotypic variances in the whole population in Env. 1 and 2, respectively, was located in the first intron of Wx (Table 1 and Figure 5D). Other SNPs in Wx showed different LD associations with sf0601764762 (Figure 5D). Wx is the most important genetic determinant of amylose content (Tian et al., 2009). We identified eight major Wx haplotypes with 23 SNPs (Figures 5E,F and Supplementary Table S6). Hap1, 6, and 7 showed lower Alb values than hap4 and 5 in both environments. The lead SNP sf061764762 is located to the first intron of Wx, the major gene determining starch content. This SNP generates alleles Wx<sup>a</sup> with a normal GT sequence at the 5 0 splice junction of intron 1, and Wx<sup>b</sup> with a G to T mutation in intron 1. Wx<sup>a</sup> and Wx<sup>b</sup> produce a mature 2.3 kb Wx mRNA and a 3.3 kb Wx pre-mRNA, respectively (Wang et al., 1995). higher Pro exhibited much higher expression levels in indica group in this study. <sup>4</sup>http://crep.ncpgr.cn/crep-cgi/home.pl signals of RP6 (E) and RM1 (G) in various tissues of ZS97 based on the microarray data. The y-axis represents the expression signals. (H) Representation of pairwise r 2 values among polymorphic sites in RP6 and RM1. The lines in red represent lead SNP. Expression levels of RP6 (I) and RM1 (J) in the endosperm at 7 days after pollination in indica group. Error bars, SE of 3 replicates. ∗∗Indicates the differences of expression levels between two haplotypes are significant at P < 0.01. Hap, haplotype; HD, heading date; DAP, day after pollination. We checked the quantity of 2.3 and 3.3 kb Wx RNA with four haplotypes in 35 and 37 accessions randomly chosen from indica and japonica, respectively. Using qRT-PCR analysis, it showed that quantity of 2.3 kb Wx RNA in the endosperm at 7 d.a.p. in hap7 indica accessions were much higher than those in hap2 indica accessions, hap3 japonica accessions and hap5 japonica accessions (P < 0.01). In contrast, quantity of 3.3 kb Wx RNA in hap7 indica accessions were much lower than those in hap2 indica accessions and in hap5 japonica accessions (Figures 5G,H and Supplementary Table S7). With highest Alb content among four haplotypes, hap5 had relatively high 3.3 kb Wx RNA quantity and low 2.3 kb Wx RNA quantity. However, with lowest Alb content among four haplotypes, hap7 had relatively low 3.3 kb Wx RNA quantity and highest 2.3 kb Wx RNA quantity. It was found that Wx exhibited higher level quantity of 2.3 kb Wx mRNA in endosperm in ZS97 (hap7) than Minghui 63 (hap2), two indica cultivars (Figure 5I). We further compared the quantity of 2.3 and 3.3 kb Wx RNA in various tissues between two varieties Zhonghua 11 (hap2) and ZS97 by quantitative RT-PCR (Figure 5J). The results showed that ZS97 had lower 3.3 kb Wx RNA quantity and higher 2.3 kb Wx RNA quantity than Zhonghua 11 in endosperm of 7 and 14 d.a.p. However, Wx displayed very low-level expressions in stem, sheath, and flag leaf in both ZS97 and Zhonghua 11. Then we measured amylose content of the corresponding accessions in 2015 (Supplementary Table S7). The correlation analysis among Alb, amylose content, quantity of 2.3 and 3.3 kb Wx RNA was performed, and the results are presented in Table 2. Amylose content had significant correlations with both quantity of 2.3 and 3.3 kb Wx RNA, but amylose content was positively correlated with quantity of 2.3 kb Wx RNA and was negatively correlated with quantity of 3.3 kb Wx RNA, which was consistent with previous study (Wang et al., 1995). Significant and negative correlations were observed between Alb and amylose content or quantity of 2.3 kb Wx RNA (Table 2), suggesting that Wx may negatively regulate Alb. However, Alb and quantity of 3.3 kb Wx RNA had no significant correlations. We speculated that Wx might influence Alb content. ### Analyses of Four Candidate Genes Association analysis at the haplotype level greatly increases mapping power (Han et al., 2016). We re-detected these grain-quality-related genes mentioned above at the haplotype level (Supplementary Table S8). Four SSP loci (OsAAT2, Alb of 46 accessions in 2015 was measured; AC, amylose content of 44 accessions in 2015; 2.3 kb, relative expression level of 2.3 kb Wx; 3.3 kb, relative expression level of 3.3 kb Wx. <sup>∗</sup>P < 0.05 and ∗∗P < 0.01. RA17, RM1, and RP6) and four starch-metabolism-related genes (AGPS2a, ISA2, PUL, and Wx) were detected in both environments; however, GBSSII, GluA1, and RA16 were only detected in one environment. The order of magnitudes of the P-values for these genes was greatly reduced compared with that at the SNP level. To further verify the association possibility, we validated some of candidate genes detected via GWAS by haplotype analyses and expression profiles in public databases. To determine whether novel functional loci were implicated by GWAS, we investigated SNP sf0515211855 (P = 6.5E-07 in Env. 1 and P = 2.7E-11 in Env. 2), in Chr.5 (14.7- 15.8 Mb) (Figures 6A–C). This lead SNP was significantly associated with Pro and explained 8.5 and 13.7% of the phenotypic variance in the whole population in Env. 1 and 2, respectively (Table 1). SNPs in the region were grouped into two LD block, and sf0515211855 was in the first block. Within this block, there were a gene cluster of 18 prolamin precursors and a candidate gene encoding an expressed protein (LOC\_Os05g25500) (Figure 6B). Within the represents the expression signals. HD, heading date; DAP, day after pollination. cluster we focused on PROLM1 (LOC\_Os05g26240), located close to the lead SNP, and identified twelve major haplotypes (Hap1-12) (Figures 6D,E and Supplementary Table S6). Hap4, 5, and 6 had significantly higher Pro than other haplotypes in both environments. Microarray data (see foot note text 4) indicated that PROLM1 had extremely high expression enrichment in endosperm of ZS97 at the ripening stage (Figure 6G). We obtained five major haplotypes (Hap1-5) for LOC\_Os05g25500 (Supplementary Table S6). Hap3 and 5 had significantly higher Pro content than Hap1 and 4 in both environments (Figure 6F). LOC\_Os05g25500 exhibited higher expression levels in endosperm than other tissues of ZS97 (Figure 6H). We focused on other two candidate genes LOC\_Os03g29750 (encoding an expressed protein) and LOC\_Os02g13130 (encoding a KH domain-containing protein) for Glo in indica group in 2015 (Figure 7A) and Glu in all group in 2014 (Figure 7B), respectively. Hap5 of LOC\_Os03g29750 had significantly higher Glo than other haplotypes, and LOC\_Os03g29750 showed very high-level expression in endosperm of ZS97 (Figure 7C). Hap4 of LOC\_Os02g13130 had significantly higher phenotypes than other haplotypes. For LOC\_Os02g13130, homologous with maize gene encoding high molecular weight glutenin subunit x, it had high-level expressions in most tissues and organs (Figure 7D). These results indicated that these genes might be good candidates for the GWAS locus. HD, heading date; DAP, day after pollination. ### DISCUSSION ### Phenotypic Variation and Trait Correlation The contents of the four components of SSP and the total SSP were normally distributed in two environments and influenced by environment in this study (Figure 1). The indica subpopulation showed wider variation in Pro than the japonica subpopulation, but narrower variation compared to japonica for Alb. These results indicated that there were probably different genetic bases underlying Alb and Pro in the two subpopulations. The contents and proportions of different components as well as total SSP in the present study were in agreement with those reported by Kumamaru et al. (1988). Glu is highest composition of SSP in rice endosperm and has more essential amino acid required for human (Tanaka et al., 1995), suggested that it is more effectively to improve protein content and nutritional value of rice by increasing Glu than other three SSPs. In previous studies, different conclusions were reached on the heritability of SSP content in rice. Hillerislambers et al. (1973) reported that the heritability of SSP content was 13.3–37.2% in rice. However, Shenoy et al. (1991) obtained values as high as 71% and considered that it is effective to select protein content phenotypes in early generation in the protein content breeding. Here, different SSP showed different heritabilities (29.5–76.8%) (Supplementary Table S3). It suggested that four SSP may have differences in generation selection. To some extent, Alb with higher heritability was effectively selected in early generation, but it may not be good for Glu and Total with lower heritability. The relationship between different SSP components in rice is complex. In the present study, there were significant positive correlations between Glu and the other three SSPs in both environments (Supplementary Table S4), that was consistent with reported study (Zhang et al., 2008). These results indicated that Glu and other three SSPs might partially share the common genetic mechanism. ### GWAS for Seed Storage Protein in Rice The use of high-density, genome-wide SNPs in GWAS not only can detect candidate genes, but also have a comprehensive understanding of the regulatory mechanism of related traits. Among the 107 associations detected in the study, few sites were repeatedly detected in both environments. Compared with Glo and Glu, Alb and Pro have larger variations in the populations (Supplementary Table S5). Similarly, association mapping was more efficient in identifying associations for Alb and Pro with relatively higher heritability than total SSP and Glu with lower heritability (Supplementary Tables S3–S5). On the other hand, we noticed that all associations detected for Alb and Pro explained more than 100% of the phenotypic variance. This suggests that phenotypic variance explained by the interaction between some of these associations might be very large. Although genome-wide association studies are becoming more sophisticated, it should be noted that association mapping may lead to false positive associations largely caused by population structure. In order to reduce false-positives resulting from genetic structure, we also analyzed the indica and japonica panels separately by LMM. Thirteen associations were adjacent to previously known grain-quality-related genes (Table 1 and Supplementary Table S5). These results suggested that association mapping was an effective way to find candidate genes for SSPs in rice. And 28 associations were detected in previously reported intervals or QTLs (Aluko et al., 2004; Wang et al., 2008). QTL pro6 was repeatedly detected for Alb (sf0605251091 and sf0601764762) and Glu (sf0602321094). Lead SNPs (sf0709447538 and sf0712842943 for Glu, sf0705739605 and sf0706196757 for Pro) were co-located in intervals 7–4 and 7–5. Although large numbers of QTLs for grain protein content were detected in the past; only one major QTL has been cloned (Peng et al., 2014). For the lead SNP sf0709447538, explaining 12.7% of the Glu variation and located to the same chromosome region as QTL (7–4 and 7–5), we developed NILs to confirm the QTL. The results showed that it was significantly difference in Glu between two NILs and suggested that the QTL was reliable. To further purify the genetic backgrounds and fine map the QTL, it is needed to backcross the NILs for recombinant screening. It has been reported that a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein has been successfully cloned by GWAS and linkage mapping analysis (Liu et al., 2016). We detected a lead SNP responsible for Alb in the Wx gene region (Figure 5 and Table 1), This region is considered a hotspot of major QTLs (6-2; qPC-6; and pro6) for protein content in rice (Aluko et al., 2004; Wang et al., 2008; Lou et al., 2009; Yu et al., 2009). Analysis of the correlation among Alb, amylose content and quantity of 2.3 kb Wx RNA showed that Wx might influence Alb content, that needs transgenic experiment to verify the function of Wx. Additionally, Starchmetabolism-related genes, such as PUL (Fujita et al., 2009), ISA2 (Utsumi et al., 2011), GBSSII (Hirose and Terao, 2004), Flo4 (Kang et al., 2005), and AGPS2a (Akihiro et al., 2005) were associated with SSPs in different populations. Overexpression of albumin gene RAG2 significantly increased total protein content, prolamin, glutelin, and amylose content, but decreased total starch (Zhou et al., 2016). Carbon and nitrogen metabolisms show a cooperative modification, and consequently, validation the function of these associations might help to better understand the genetic relationship between SSP and starch contents in rice grain. ### Application in the Improvement of Grain Quality in Rice Grain with good quality could be developed by regulating the SSP content. SSP is a typical quantitative trait typically affected by environment (Shewry, 2007). Combination of conventional breeding and molecular techniques, e.g., markerassisted selection (MAS), may provide a more efficient approach for improving the SSP content of the grain than classical breeding alone (Zhang et al., 2008). The Lgc1 mutation has been used to development new low easy-to-digest protein rice varieties with low glutelin content and high prolamin content, which is useful for patients with chronic renal failure (Mochizuki and Hara, 2000; Nishimura et al., 2005; Morita et al., 2009). O2 mutation could increase free lysine and tryptophan levels by reducing the synthesis of zeins in maize, which is useful for QPM breeding (Mertz et al., 1964; Liu et al., 2016). Genes and possible causative SNPs identified in the present study could be used as potential targets for rice grain nutritional quality improvement. Glutelin is the most easily to digest and contains high lysine (Tanaka et al., 1995). The lead SNP sf0709447538 co-located in QTLs (7–4, 7–5, and 7–6) was validate to have an effect on glutelin, which could be targets for map-based cloning of the candidate genes to illuminate the molecular mechanism of glutelin and improve grain nutritional quality by MAS. RM1 and RP6 are adjacent genes on chromosome 7, and with higher levels expression and higher Pro, hap2 of RP6 and hap3 of RM1 are distributed mainly in the indica subpopulations (Figure 4 and Supplementary Table S6). Therefore, the region covering RM1 and RP6 from japonica subpopulation can be a promising target for reducing Pro content and achieving better nutritional quality in indica cultivars. Wx may be a key gene for regulating the content of Alb and amylose content. The availability of the Wx gene sequence provides the possibility of improving the protein content via Wx gene modification. Different haplotype combinations of candidate genes for SSP would produce grains with different eating and nutritional quality. Genes and possible causative SNPs identified in the present study could be useful for breeding rice cultivars with favorable eating and nutritional quality. ### CONCLUSION In the present study many associations were identified for five SSP traits using GWAS in two environments. We detected novel associations, known SSP genes, and SNPs adjacent to known starch-metabolism-related genes. We also analyzed haplotypes of known grain-quality-related genes. Our results suggested that GWAS was an effective way to identify genes for rice SSP traits and the level of 3.3 kb Wx pre-mRNA is positively correlated with Alb content, providing new insights into the genetic basis of rice quality. Overall, we provided useful information that could be used in future gene functional studies and rice quality improvement. ### AUTHOR CONTRIBUTIONS YH designed and supervised the experiments. PC, ZS, GL, and BW performed all the phenotypic evaluations. LM, YiL, PC, WD, and BP performed analysis and interpretation of the data. PC wrote the paper. GW, YaL, and DZ provided rice germplasm samples. GG, QZ, JX, and XL participated in the field management and logistic work. ### FUNDING This work was supported by grants from the from the Ministry of Science and Technology of China (Grants ### REFERENCES 2016YFD0100501, the National Program on R&D of Transgenic Plants (2016ZX08009-003-004 and 2016ZX08001002-002), the National 863 Project (2014AA10A604), and the earmarked fund for the China Agriculture Research System (CARS-01-03) of China. ### ACKNOWLEDGMENTS We thank Xufeng Bai, Peng Yun, Qiuxiang Luo, Haijiao Dong, Hao Zhou, Pingbo Li, Quanxiu Wang, Dujun Wang, Yuanyuan Zheng, Zhongmin Han, Xiaokai Li, Hu Zhao, and Wei Chen for editing, suggestions, and assistance. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00612/ full#supplementary-material (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm. J. Exp. Bot. 60, 1009–1023. doi: 10.1093/jxb/ern349 amylopectin biosynthesis in rice endosperm. Plant Physiol. 156, 61–77. doi: 10.1104/pp.111.173435 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Chen, Shen, Ming, Li, Dan, Lou, Peng, Wu, Li, Zhao, Gao, Zhang, Xiao, Li, Wang and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Banana21: From Gene Discovery to Deregulated Golden Bananas Jean-Yves Paul<sup>1</sup> \, Robert Harding<sup>1</sup> , Wilberforce Tushemereirwe<sup>2</sup> and James Dale<sup>1</sup> <sup>1</sup> Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD, Australia, <sup>2</sup> National Agricultural Research Organisation, Kampala, Uganda Uganda is a tropical country with a population in excess of 30 million, >80% of whom live in rural areas. Bananas (Musa spp.) are the staple food of Uganda with the East African Highland banana, a cooking banana, the primary starch source. Unfortunately, these bananas are low in pro-vitamin A (PVA) and iron and, as a result, banana-based diets are low in these micronutrients which results in very high levels of inadequate nutrition. This inadequate nutrition manifests as high levels of vitamin A deficiency, iron deficiency anemia, and stunting in children. A project known as Banana21 commenced in 2005 to alleviate micronutrient deficiencies in Uganda and surrounding countries through the generation of farmer- and consumer-acceptable edible bananas with significantly increased fruit levels of PVA and iron. A genetic modification approach was adopted since bananas are recalcitrant to conventional breeding. In this review, we focus on the PVA-biofortification component of the Banana21 project and describe the proof-of-concept studies conducted in Australia, the transfer of the technology to our Ugandan collaborators, and the successful implementation of the strategy into the field in Uganda. The many challenges encountered and the potential future obstacles to the practical exploitation of PVA-enhanced bananas in Uganda are discussed. Keywords: East African highland banana, staple crop, Uganda, micronutrient deficiency, vitamin A deficiency, pro-vitamin A, carotenoids, biofortification ### INTRODUCTION Vitamin A (VA) or retinol is an important nutrient which supports vital physiological and developmental functions. Since it cannot be synthesized de novo, VA has to be acquired through a diversified diet (Fraser and Bramley, 2004; Kimura et al., 2007; Beyer, 2010; Fitzpatrick et al., 2012). Whereas retinol is derived directly from animal sources, plant-derived pro-vitamin A carotenoids (PVACs) such as α- and β-carotene must first be converted into retinol by the body (van den Berg et al., 2000). The majority of populations living in developing countries depend on starchy food staples such as cassava (Manihot esculenta), maize (Zea mays), potato (Solanum tuberosum), rice (Oryza spp.), plantain, and banana (Musa spp.) which unfortunately are largely deficient in essential micronutrients such as PVACs. Vitamin A deficiency (VAD) causes a number of VAD disorders including night and total blindness, premature death (Sommer and Vyas, 2012), and reduced immunity leading to increased risk of childhood infections and high infant mortality (Herbers, 2003; Bai et al., 2011). VAD affects an estimated 190 million pre-school children worldwide, most of whom live in developing countries, with a reported 5.17 million registered cases of clinical or severe levels of VAD (WHO, 2009). It is also estimated that 250,000–500,000 children become blind due to VAD each year, ### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil ### Reviewed by: Michael A. Grusak, Children's Nutrition Research Center, United States Hamid Khazaei, University of Saskatchewan, Canada > \Correspondence: Jean-Yves Paul [email protected] ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 13 February 2018 Accepted: 09 April 2018 Published: 26 April 2018 ### Citation: Paul J-Y, Harding R, Tushemereirwe W and Dale J (2018) Banana21: From Gene Discovery to Deregulated Golden Bananas. Front. Plant Sci. 9:558. doi: 10.3389/fpls.2018.00558 170 half of whom die within 12 months of losing their sight (WHO, 2009; Barber et al., 2012). In Uganda, 20% of children aged 6 months to 5 years and 19% of women aged 15–49 years suffered from VAD in 2006, predominantly in the low-income demographics within central Ugandan communities, which heavily rely on banana as a staple food (UDHS, 2006). In the Great Lakes region of East Africa highland bananas (EAHBs) are an important food security crop and the main food staple (Adeniji et al., 2010). In Uganda, it is estimated that 75% of farmers grow bananas contributing to about 7% of global banana and plantain production (Ssebuliba et al., 2006). In addition, Uganda is the largest banana consumer in the world with an estimated per capita consumption of 220–250 kg/year (Tushemereirwe et al., 2006). In rural populations of Uganda where EAHBs form a major and sometime unique part of the diet, 20 µg/g dry weight (dw) β-carotene equivalent (β-CE) is the minimum amount of PVA required in banana fruit to provide 50% of the estimated average requirement (EAR) of VA (Paul et al., 2017). Despite their considerable biodiversity, most EAHB varieties grown in East Africa have low levels of essential micronutrients such as iron (Fe), zinc (Zn), and PVACs (Davey et al., 2009; Fungo et al., 2010). Overreliance on banana in this region, and in Uganda in particular, has contributed to the exacerbation of micronutrient deficiency-related illnesses. Strategies such as diet diversification, supplementation, and food fortification have been used to help alleviate some of these ailments with varying levels of success (WHO, 2009; Gómez-Galera et al., 2010; Fitzpatrick et al., 2012; Bruins and Kraemer, 2013). However, whereas these interventions are very successful in an urban context where the target population is in the vicinity of service providers, they fail to reach rural communities that are most in need (Dalmiya and Palmer, 2007; Victora et al., 2008; WHO, 2009). As such, in Uganda, VADs in children are relatively low in urban areas but remain elevated in rural communities (UDHS, 2006). More recently, biofortification has emerged as a complementary, cost-effective, and sustainable approach to deliver micronutrient-dense crops to the poorest and hardestto-reach communities. This can be achieved through either conventional breeding, where the necessary traits are available within the accessible "breeder's gene pool," or through genetic modification. Examples of successful conventional breeding approaches include the biofortification of maize (Egesel et al., 2003; Harjes et al., 2008), sweet potato (Mwanga et al., 2009), and cassava (Ceballos et al., 2012). These biofortified products are already being disseminated in various parts of the world including Uganda (Anderson et al., 2007; Hotz et al., 2012; Talsma et al., 2016). Arguably the most successful example of biofortification through genetic engineering is the development of Golden Rice (GR) (Ye et al., 2000; Paine et al., 2005). Considering the popularity of EAHBs in East Africa, biofortification of this food staple with enhanced levels of PVACs (or other micronutrients) is now believed to be the best long-term, sustainable, and cost-effective strategy to ease the burden of VAD in high risk populations of Uganda. The use of conventional breeding to develop PVA-biofortified EAHBs is constrained by their low fertility and the lack of high PVA EAHB varieties in the known gene pool. Further, any new varieties developed are unlikely to possess the attributes of locally preferred landraces of EAHBs. Genetic modification is therefore the fastest and most reliable approach to improve the existing preferred varieties. Here, we review the foundations, goals, achievements, future prospects, and challenges of Banana21<sup>1</sup> , a project that undertook the challenges of alleviating micronutrient deficiencies in Uganda by enhancing the nutritional content of its staple food, EAHBs, through genetic modification. ### THE PROMISE OF BANANA21 – MORE NUTRITIOUS BANANAS MADE IN UGANDA, BY UGANDAN SCIENTISTS, FOR THE UGANDAN PEOPLE Banana21 is one of four original projects funded by the Grand Challenges in Global Health (GCGH) program of the Bill and Melinda Gates Foundation (BMGF) to "Create a Full Range of Optimal, Bioavailable Nutrients in a Single Staple Plant Species." Banana21 is a collaborative research project between the Centre for Tropical Crops and Biocommodities at Queensland University of Technology (QUT) in Australia and the National Banana Research Program of the National Agricultural Research Organisation (NARO) of Uganda. The project aims to help "Alleviate VAD and iron deficiency anemia through the micronutrient enhancement of the staple food of Uganda, bananas." From the outset, it was recognized that the success of the project depended on its objectives being widely understood and accepted in Uganda. As such, we considered it extremely important to train and empower young Ugandan scientists to deliver the project milestones thus ensuring the biofortified genetically modified (GM) bananas were generated in Uganda, by Ugandan scientists, from Ugandan varieties, and for the benefit of the Ugandan people. As a consequence, in addition to scientific discoveries and their applications, Banana21 has been a capacity building project where technology transfer continues to be at the core of every phase, milestone, and decision-making activity. An overview of the Banana21 project strategy is shown in Figure 1. Initially, the proof-of-concept research was developed at QUT using the locally grown dessert banana cultivars "Cavendish." The QUT components included gene and promoter discovery, tissue culture and transformation, field trials of GM bananas, and downstream fruit sampling and analysis. The development and implementation of a comprehensive stewardship plan was also a key project component. Following proof-of-concept in Australia, the technology and know-how was to be continuously transferred to NARO in Uganda to generate PVA-biofortified local EAHB varieties. Further, it was necessary for the infrastructure to be implemented at NARO to ensure that the GM plants produced would be tested in the laboratory and the field with the consistency and rigor necessary to generate the data required for a GM product deregulation dossier. <sup>1</sup>http://www.banana21.org/ ### THE CHALLENGES AND ACHIEVEMENTS OF BANANA21 – TRANSGENIC BIOFORTIFIED EAHBs WITH SIGNIFICANTLY HIGHER FRUIT PVA CONTENT ### Phase 1 – Early Discovery and Technology Transfer HDR, higher degree research. At the commencement of the project in 2005, the only practical demonstration of PVA biofortification in a staple food crop was GR. The initial GR strategy involved the re-engineering of a carotenoid biosynthesis pathway in normally carotenoid-free rice endosperm by the endosperm-specific [glutelin 1 (gt1) promoter] expression of a daffodil-derived phytoene synthase (psy) transgene in combination with the constitutive (CaMV 35S) expression of a bacterial phytoene desaturase (crtI) gene. Although successful, the level of carotenoid accumulation in rice endosperm was still considered to be too low for practical exploitation (Ye et al., 2000). Subsequent research revealed that the origin of the psy transgene and choice of promoter were important factors affecting PVAC accumulation levels (Paine et al., 2005). This led to the development of Golden Rice 2 (GR2) whereby the use of a maize-derived psy transgene and bacterial crtI, both under the control of the gt1 promoter, resulted in high levels of PVAC accumulation (Paine et al., 2005). Based on this success, the GR2 strategy was deemed to be the most logical approach to develop VA-biofortified bananas. One of the major initial challenges was the identification of suitable transgenes and promoters for expression in banana. As such, the early phase of the project focused on designing and testing large numbers of expression constructs containing a suite of different promoters and transgene combinations. Due to the paucity of information regarding transgene expression in banana fruit, it was necessary to assess a range of different promoters for their spatiotemporal activity in banana, particularly fruit. As such, several constitutive promoters were isolated from different sources in addition to promoters that controlled the expression of genes involved in banana fruit development. These were fused to the β-glucuronidase reporter gene (uidA), transformed into banana embryogenic cell suspensions (ECS) (Khanna et al., 2004) and transgenic plantlets regenerated. A list of all promoters tested, their origin, and specificity is presented in Table 1. In combination/parallel with the promoter characterization study, a range of different transgenes were also assessed. Initially, the strategy used to develop GR2 was adapted to banana and the first generation of expression constructs was made to express the maize phytoene synthase 1 (ZmPsy1B73) transgene alone or in combination with the bacterial (Pantoea ananatis) carotene desaturase transgene (PaCrtI) and controlled by various promoters (Table 1). Our efforts also focused on isolating and using PVA-associated cisgenes from banana in the hope of minimizing gene silencing and thus achieving more stable gene expression over several generation. The use of banana-derived cisgenes/s was also considered to be more advantageous from a future deregulation perspective. The biodiversity of banana and plantain is huge especially in Southeast Asia and Papua New Guinea where bananas are believed to have originated (Perrier et al., 2011). As such, the ability of various cultivar to accumulate PVACs in their fruit is also very diverse (Davey et al., 2009; Ekesa et al., 2015). Of particularly interest to us was a small group of bananas originating from the Pacific called the Fe'i bananas (Musa troglodytarum) which accumulate extremely high levels of fruit PVACs (Englberger et al., 2003). One such Fe'i banana variety, "Asupina," became our model cultivar for not only understanding carotenoid metabolism and accumulation in banana fruit but also as the source of a cisgene encoding a phytoene synthase, MtPsy2a (Mlalazi et al., 2012; Buah et al., 2016). Following the cloning and molecular characterization of MtPsy2a, expression cassettes containing this gene with and without PaCrtI and controlled by various promoters were also constructed and used to generate transgenic "Cavendish" banana lines in order to assess the levels of PVA accumulation in fruit. During Phase 1, an important training program was also initiated whereby several students from Uganda and Kenya commenced their higher degree research (HDR) doctoral studies at QUT. The research projects developed for these students focused primarily around banana tissue culture and transformation technologies so that the knowledge and experience gained in the QUT laboratories would be transferred back to Africa for the benefit of the project. Simultaneously, TABLE 1 \| List of promoters and genes tested in AFT-1 of the Banana21 project. training of technical staff and capacity building around infrastructure and laboratory equipment began at NARO, Uganda. A major component of this training program was the development of protocols and standard operating procedure (SOP) for generating and transforming ECS of local banana varieties. From the beginning, the Ugandan component of the project focused on establishing ECSs from three popular Ugandan banana cultivars, namely "Nakitembe," "M9," and "Sukali Ndiizi" since these were considered the most appropriate target cultivars for biofortification. True EAHB varieties such as "Nakitembe" are the preferred cultural choice among Ugandans and are usually grown in the highlands where disease pressure is minimal. The disease-resistant EAHB hybrid "M9" is less popular but was chosen as it is more productive in the lowlands where diseases such as black Sigatoka are a serious limitation to banana production. "Sukali Ndiizi" was chosen because it is a small sweet banana which is popular among children, the most vulnerable target population. ### Phase 2 – Proof of Concept, Field Trials in Australia and Uganda The banana fruit PVAC target levels necessary to deliver 50% of the EAR of VA in vulnerable populations was estimated at 20 µg/g dw β-CE. The PVA-biofortification proof-of-concept research was done in Australia using transgenic "Cavendish" and "Lady Finger" bananas as the models with the aim of obtaining the target fruit PVA levels with no changes in agronomical characteristics. One of the major limitations of the project was the lengthy timeframe from transformation of banana ECS through to fruit harvest which is approximately 30 months. This limitation precluded the serial testing of our expression constructs. Consequently, our strategy was to transform "Cavendish" and "Lady Finger" ECS in Australia with a large number of different expression constructs, regenerate transformants, and field trial all the plants in parallel. At the end of Phase 1, between 10 and 30 independents transgenic "Cavendish" banana lines for each of the expression constructs had been generated. Considering the very large number of lines to be tested, and the fact that bananas are a very large crop, it was not practical to conduct a trial in the glasshouse. With these limitations in mind, we assessed these lines directly in the field without prior glasshouse characterization. The first Australian field trial (AFT-1) of GM "Cavendish" banana lines was subsequently established in 2009. This trial contained all available independent lines for each construct but only a single plant per line. A total of 28 constructs were tested, 14 to test promoter activity (as promoter/uidA reporter gene fusions) and 14 to test the same promoters in combinations with three transgenes (MtPsy2a, ZmPsy1B73, and PaCrtI) encoding proteins involved in the biosynthesis of PVACs (Table 1). This was the first GM banana field trial in Australia and was conducted under a license issued by the Australian Office of the Gene Technology Regulator (OGTR)<sup>2</sup> . Of the 14 promoter/uidA fusion combinations tested in the field, three promoters (Ubi, Exp1, and Aco) consistently conferred the strongest levels of GUS expression. The constitutive maize polyubiquitin (Ubi) promoter showed consistently strong activity from the earliest stages of fruit development through to maturity. In contrast, the banana fruitspecific expansin 1 (Exp1) and ACC oxidase (Aco) promoters were only active in the later stages of fruit development (Paul et al., 2017). When we subsequently investigated the accumulation of PVACs during fruit development, a similar trend was seen whereby constitutive expression of either <sup>2</sup>http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir076rarmp-toc~ dir076rarmp-ch1~dir076rarmp-ch1s5 MtPsy2a or ZmPsy1B73 using the Ubi promoter increased PVA accumulation from the earliest stages of fruit development while PVAC accumulation was restricted to the late stages of fruit development when the same transgenes were tested under the control of either the Exp1 or Aco promoters. Analysis of fruit samples from plants transformed with MtPsy2a, ZmPsy1B73, and/or PaCrtI revealed that constitutive (Ubi) expression of MtPsy2a resulted in the highest fruit β-CE levels which reached almost 19 µg/g dw in the plant crop (Figure 2A). Although this was just below the target level, it nonetheless represented a 11-fold increase over wild-type banana PVA baseline levels and was highly encouraging (Paul et al., 2017). A second Australian field trial (AFT-2) was planted in September 2012 to test the efficacy of new PVA biofortification constructs as well as to monitor transgene stability in multiple plants of selected lines from AFT-1 through three successive generations. The additional PVA genes and promoters tested in this second field trial (Table 2) were tested in combination with the MtPsy2a gene under the control of various promoters. Unfortunately, none of the new combinations tested were found to elevate fruit PVA levels as high as those achieved by the constitutive expression of MtPsy2a alone in AFT-1. However, an extremely interesting and important outcome from AFT-2 was the observation that fruit PVA levels increased over successive generations in some lines to levels exceeding the target. For example, the fruit PVA levels of a line containing Ubi-MtPsy2a was found to increase from 11.7 µg/g dw β-CE in the first generation to 75.1 µg/g dw β-CE in the fourth generation. Qualitative and quantitative phenotypic and agronomical data have been recorded for all the lines tested and has revealed some interesting trends (Paul et al., 2017). The origin of the transgene had the biggest impact with, for example, banana lines expressing the banana MtPsy2a appearing normal with no variation in critical agronomical features such as yield and cycle time. In contrast, although expression of the maize Psy1B73 gene resulted in increased fruit PVA levels, many lines with undesirable phenotypes such as stunting and photo bleaching were observed. During Phase 2, scientists at NARO had established regenerable ECSs, as well as efficient transformation protocols, for two varieties, "M9" and "Sukali Ndiizi." These breakthroughs allowed the generation of transgenic banana plants from both varieties containing either the ZmPsy1B73 or the "Asupina" derived phytoene synthase 2a (MtPsy2a) transgenes. Since the results of AFT-1 were not available when this activity commenced, these gene were placed under the control of the banana-derived Exp1 promoter (Mbabazi, 2015). The transgenic lines generated from both cultivars were subsequently grown in a field trial at NARL-NARO, Kawanda Uganda in 2010 to assess the fruit PVAC levels. This first Ugandan field trial (UFT-1), conducted under authorization from the Ugandan National Biosafety Committee (NBC), was a very important milestone for Banana21 as it represented the first GM crop field trial in sub-Saharan Africa where the events had been created by local scientists from a national laboratory. At the completion of Phase 2, proof-of-concept for PVA biofortification had been demonstrated in Australia using "Cavendish" and "Lady Finger" bananas and revealed that target fruit PVA levels could be achieved using the constitutive (Ubi) or fruit-specific (Aco) expression of MtPsy2a alone (Paul et al., 2017). Further, very few off-type traits, such as reduced yield and increased cycle time, were observed in the transgenic lines and the trait appeared stable over successive generations. Importantly, the field trial in Uganda (UFT-1) produced multiple lines with fruit PVA levels higher than their respective controls including one Exp1-MtPsy2a line of M9 with 33.1 µg/g dw β-CE (Mbabazi, 2015). With this knowledge, a new generation of plant expression vectors were made at QUT and two constructs containing Ubi-MtPsy2a and Aco-MtPsy2a FIGURE 2 \| (A) PVA-biofortified "Cavendish" banana in Australia and (B) PVA-biofortified (left) and wild-type control (right) "Nakitembe" EAHB in Uganda. TABLE 2 \| List of new promoters and genes tested in AFT-2 of the Banana21 project. were transferred to NARO for transformation into EAHB varieties. ### Phase 3 – Product Development: Early Events Selection Field Trial in Uganda This phase of the project (2012–2017) initially involved generating a total of 200 independent transgenic lines each of EAHB cultivars "M9" and "Nakitembe" containing MtPsy2a under the control of either the Ubi or the Aco promoters. Following molecular characterization by the now fully trained technical staff and highly qualified scientists at NARO, the transgenic lines were field planted (UFT-2) at NARL-NARO, Kawanda in August 2014 to identify suitable elite lines for further testing in multi-location field trials (MLTs). During those 5 years, the NARO team showed exceptional professionalism in learning and implementing good practices around generating, handling, and tracking GM products. The results from UFT-2, which will soon be published, clearly demonstrate that fruit PVACs can consistently accumulate at levels above the required target of 20 µg/g dw β-CE in GM-biofortified "M9" and "Nakitembe" without phenotypic alteration of the plants (Figure 2B). From the current transgenic line selection trial at Kawanda, 10 elite lines each of "M9" and "Nakitembe" will be selected to progress through to future MLTs. The initial selection process began in 2017 by selecting lines with fruit PVA levels equal or greater than 20 µg/g dw β-CE at the full green developmental stage (harvesting stage in Uganda) and yield within 20% of non-GM controls plants. From this initial selection, molecular analysis was used to identify lines containing fewer than three copies of the integrated expression cassettes with a preference for single integrations. Although selection of "single copy" events is preferred in seed crops to produce homozygous lines that do not segregate for the transgenic trait in future generations, in a vegetatively propagated crop such as banana, it is only rationalized to increase the likelihood of events with "clean insert." During this phase, NARO scientists also conducted a preliminary blind sensory panel test of traditionally prepared banana meal (matooke) using fruit from both non-GM and PVA-biofortified "M9" and "Nakitembe" to compare appearance and texture but not taste. Interestingly, 80% of the panelists (n = 15) rated fruit from one of the "M9" PVA-biofortified lines as the most preferred whereas fruit from its non-GM counterpart had the highest dislike proportion (29%). On the basis of these encouraging results, Banana21 entered its fourth and final phase of funding in October 2017. This final phase is focused on generating all the data required for the deregulation of a GM PVA-biofortified EAHB in Uganda. The prospect of the release of the world's first deregulated GM banana developed from an African laboratory is as much exciting as it is daunting. ### THE FUTURE CHALLENGES OF BANANA21 – THE NOT SO LONG ROAD AHEAD TO DEREGULATION From a technology perspective, the groundwork in Uganda has been completed and the exceptional results from UFT-2 have provided the ideal platform to further select elite lines to progress through to MLTs and ultimately to farmer release. Prior to MLTs, critical data necessary for the compilation of a deregulation dossier must be obtained. Whole-genome sequencing of the pre-selected lines is necessary to allow the site(s) of transgene insertion into the host genome to be identified. This is necessary to ensure that (i) the transgene is intact, (ii) there are no new open-reading frames created, (iii) there is no disruption of endogenous open-reading frames, and (iv) no plasmid sequence has been integrated into the host genome. Finally, it is essential that the composition of the fruit from the deregulated lines is very similar to that derived from the wild type. Therefore, compositional analysis for food characteristics such as calories, calories from fat, carbohydrates, protein, ash, and moisture will be done, and lines with values >15% different to controls will be discarded. The next phase of the project involves the identification of two lines (one lead and one reserve) each of "M9" and "Nakitembe" from the MLTs, and obtaining the necessary agronomic, biochemical, and molecular data to ultimately prepare the dossier for submission to regulators in Uganda for deregulation and general release. At the end of this phase of the project (December 2021), these four lines are expected to meet all the agronomic, biochemical, and molecular analyses and biosafety assessment required for deregulation in Uganda under the proposed Biosafety Bill. One of the major hurdles for Banana21 is the lack of a regulatory framework for biotechnology in Uganda. Without a regulatory body controlling the safe application of biotechnologies, Banana21 will not be able to release PVA-biofortified "Golden bananas" to farmers and consumers in need. After ratifying the Cartagena Protocol on Biosafety in 2002, it took 6 years for Uganda to approve a policy on Biotechnology and Biosafety in 2008. Under the current policy, the NBC supervises all GMO activities up to the stage of Confined Field Trial (CFT) under the supervision of the National Council of Science and Technology (UNCST) Act 1990. Therefore in 2012, the National Biotechnology and Biosafety Bill, 2012, was introduced into parliament to provide a regulatory framework and guide the implementation of modern biotechnology in Uganda to minimize any potential risks to the environment, human, and animal health. After 5 years, Uganda's Parliament passed the National Biotechnology and Biosafety Bill into law becoming the Biosafety Act, 2017 on October 4, 2017. The new law will not only benefit Banana21 but also a multitude of other biotechnology products developed in Uganda such as bananas with bacterial wilt resistance, drought tolerant maize, bollworm resistance and herbicide tolerant cotton, and new cassava varieties with resistance to Cassava mosaic and brown streak viruses. Our initial target of 20 µg/g dw β-CE was calculated using a bioconversion factor of PVACs to retinol of 6:1 based on the results of a study using Mongolian gerbils (Bresnahan et al., 2012). Underestimating this bioconversion factor would raise the target above its current value. For this reason, Banana21 with financial help form HarvestPlus and the BMGF, commissioned a nutrition study at Iowa State University to determine a more accurate bioconversion ratio in humans. Although the results from this study are not yet available, some of the PVA-biofortified lines that have been developed under Banana21 have over four-times the initial target value with no yield penalty and could potentially be substituted if all other deregulation criteria are met. The development of robust diagnostics for banana viruses was another important component of this project. These diagnostics form the basis of a banana virus indexing protocol that has been rolled out to ensure that the plantlets derived from the Banana21 project are virus tested, thus reducing the potential distribution of infected planting material to farmers. Since the majority of banana growers in Uganda are subsistence farmers, the distribution strategy adopted in the future will need to minimize the cost of planting material while maximizing the rate of distribution. Initial propagation of the lines to be released will be done at NARO and the plantlets will be sent to small banana micropropagation laboratories and also used to establish small "mother gardens" for the initial production of suckers. A cost-effective and self-sustaining strategy for dissemination will then involve identifying "innovative farmers" that will be given suckers. For every sucker, they will be asked to give away two suckers to neighbors who in turn will be asked to give away two suckers for each one received under the scheme. ### REFERENCES Adeniji, T. A., Tenkouano, A., Ezurike, J. N., Ariyo, C. O., and Vroh-Bi, I. (2010). Value-adding post harvest processing of cooking bananas A key component of the next phase of the project will be the implementation of a comprehensive stewardship and communication plan. This includes (i) forming a Technical Advisory Committee (TAC) that meets regularly and provides scientific, strategic, and biosafety expertise, (ii) implementing and regularly updating SOPs, (iii) keeping accurate and safe records of the data with tools such as the BananaTracker software developed by QUT, and (iv) meetings with the Australian OGTR and Food Standards Australia and New Zealand (FSANZ) to seek advice on the requirements for deregulation if these lines were to be deregulated in Australia. The NARO team has also been involved in various communication activities in an attempt to educate the public and de-mystify the use of GMOs. Important stakeholders are targeted through workshops and information sessions, as well as various paper-based and audio-visual communication materials. ### FINAL REMARKS AND CONCLUSION From the outset in 2005, Banana21 has been on a trajectory to develop lines of EAHBs with levels of fruit PVACs that would provide 50% of the EAR of VA with consumption of only 300 g per person per day. Based on the significant progress thus far, it is highly likely that the transgenic lines developed under Banana21 will be released by 2021 and have a significant impact in alleviating VAD in a sustainable way, especially in rural Uganda where bananas are a fundamental part of the culture. The PVA-enhanced, disease-resistant "M9" line will have the greatest impact in lower elevations of Uganda where the disease pressure is high, while the PVA-enhanced "Nakitembe" line will have greatest impact in the highlands where there is much lower disease pressure. The importance of banana as a food security crop (perennial nature, year-round production, and ability to cope with long periods of drought) associated with a low cost, farmer-driven distribution strategy should ultimately see "Golden bananas" adopted as a widespread and efficient VAD alleviating strategy in the next decade. ### AUTHOR CONTRIBUTIONS J-YP and RH drafted the initial manuscript while WT and JD reviewed and provided the constructive criticisms. ### FUNDING The Banana21 team project was, and still is, supported by a Grant from the Bill & Melinda Gates Foundation and the Department for International Development (United Kingdom). Anderson, P., Kapinga, R., Zhang, D., and Hermann, M. (2007). "Vitamin A for Africa (VITAA): an entry point for promoting orange-fleshed sweetpotato to <sup>(</sup>Musa spp. AAB and ABB genome groups). Afr. J. Biotechnol. 9, 9135–9141. combat vitamin A-deficiency in sub-Saharan Africa," in Proceedings of the 13th ISTRC Symposium, Apartado, 711–720. maize for plant breeding trials. Food Chem. 100, 1734–1746. doi: 10.1016/j. foodchem.2005.10.020 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Paul, Harding, Tushemereirwe and Dale. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. fpls-09-00558 April 24, 2018 Time: 17:15 # 8 # Improving the Yield and Nutritional Quality of Forage Crops Nicola M. Capstaff\* and Anthony J. Miller\* John Innes Centre, Norwich, United Kingdom Despite being some of the most important crops globally, there has been limited research on forages when compared with cereals, fruits, and vegetables. This review summarizes the literature highlighting the significance of forage crops, the current improvements and some of future directions for improving yield and nutritional quality. We make the point that the knowledge obtained from model plant and grain crops can be applied to forage crops. The timely development of genomics and bioinformatics together with genome editing techniques offer great scope to improve forage crops. Given the social, environmental and economic importance of forage across the globe and especially in poorer countries, this opportunity has enormous potential to improve food security and political stability. #### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil ### Reviewed by: Zhipeng Liu, Lanzhou University, China Giovanna Attene, University of Sassari, Italy #### \Correspondence: Nicola M. Capstaff [email protected] Anthony J. Miller [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 06 December 2017 Accepted: 06 April 2018 Published: 24 April 2018 #### Citation: Capstaff NM and Miller AJ (2018) Improving the Yield and Nutritional Quality of Forage Crops. Front. Plant Sci. 9:535. doi: 10.3389/fpls.2018.00535 Keywords: forage, nutritional enhancement, grass production, legumes, breeding, management ## INTRODUCTION Forage grasslands are used to feed livestock and globally it has been estimated that they represent 26% of the land area, and 70% of agricultural area (FAO, 2010). Such crops are significant economically, as the European example shows (see Figure 1). Forage crops are usually grasses (Poaceae) or herbaceous legumes (Fabaceae). Some tree legumes such as mulga (Acacia aneura) and leadtree (Leucaena leucocephala) are also grown in desert and tropical grasslands (Muir et al., 2011). In the tropics, popular grasses include Napier grass (Pennisetum purpureum), Brachiaria, and Panicum species. In the poorest parts of the world livestock production is critically important for smallholders' livelihoods. Sub-Saharan Africa is an example and frequently women maintain the livestock production systems (Njuki and Sanginga, 2013). In temperate climates, the main grasses include bentgrass (Agrostis spp.), fescue (Festuca spp.), ryegrass (Lolium spp.) and orchard grass (Dactylis spp.) or hybrids of these. For example, Festuca and Lolium hybrids has been developed from 1970s (Ghesquière et al., 2010) giving rise to crops such as Festulolium pabulare which combines the superior forage quality of Lolium multiflorum with the persistence and stress tolerance of Festuca arundinacea. Some maize (Zea mays) cultivars have been specifically bred for forage. The commonly cultivated herbaceous legumes are trefoil (Lotus corniculatus), medics (Medicago spp.), clover (Trifolium spp.) and vetches (Vicia spp.). Brassica forage species include cultivars of oilseed rape (Brassica napus) and kale (Brassica oleracea). Fodder beet (Beta vulgaris) is another temperate forage. The combination of forage crops grown in any country varies depending on climate and livestock needs, however, the perennial legume lucerne or alfalfa (Medicago sativa) is the most widely cultivated as it can be grown with both temperate and tropical grasses, or as a standalone crop. This is a huge topic to review as there are so many species grown across the world, therefore we have chosen to focus on a few examples, the tropical grasses Pennisetum and Brachiaria, and more prominently the temperate crops Lolium and alfalfa. In an ideal world, we would all eat pulses rather than the animal products generated from them, as grain legumes are the food that offers the most sustainable future (Foyer et al., 2016). There is continued pressure from many groups to lower human consumption of animal products due to livestock efficiency issues and for human health (Cramer et al., 2017). There is a lack of reliable statistics for the proportion of adults adopting a plant-based diet, but it is estimated to be between 1 and 10% of the population in developed western countries such as within the European and United States (Mcevoy and Woodside, 2010) and studies support these diets as healthy and nutritionally adequate (American Dietetic Association and Dietitians of Canada, 2003). However, the consumption of livestock products can be regarded as important to a healthy diet due to their high nutrient density (CAST, 2013) regardless of the numerous efficiency and environment concerns (Di Paola et al., 2017), particularly true in developing countries where undernourishment incidences are estimated as ∼4–22% of the population (Alexandratos et al., 2006). Livestock production can convert non-edible crops such as the forages into human food, with sustainable intensification possible when inputs and outputs of the system are balanced (Derner et al., 2017). Moreover, the cultural and social significance of livestock cannot be underestimated and the trend of increased global production is set to continue (Thornton, 2010). Livestock feature prominently across all cultures both in cuisine, but also music and literature. Additionally, in many developing countries the rearing of livestock such as cattle and goats are vital in times of hardship; many view animals as living 'piggy-banks,' that can for example pay the family school fees (Herrero et al., 2013). Therefore, in practice livestock production is set to continue throughout the world and forage crops will be grown for coming decades. Plant research has chiefly focussed on grain crops, but here we argue that there is enormous potential for improving forages. Improving the yield and nutritional quality of forage crops can help mitigate the unsustainable negative impacts of livestock production. ### FORAGE CROPS IN LIVESTOCK DIETS Forage crops can be feed directly to livestock or can be processed by partial drying or pre-digestion. Because of this processing, animal feeds can be categorized as either bulky feeds or concentrates. Bulky feeds are also termed forage and are produced from grass, cereal and legume cropping as described above, such as alfalfa, Lolium or a mixture of the two. This forage can be provided to animals directly through grazing pasture land or in a processed form, such as hay (where water content is >15%) or dried (pelleted) biomass. Concentrates are generally cereal, oilseed and legumes seeds, or bi-products of their preparation for human food, biofuel and textile. They can also include high energy feedstuffs such as sugar-rich crop molasses and fats of animal origin, for example fish by-catch discards. In industrialized countries, production of both these categories of feed can surpass the amount produced for plant-based food for human consumption; in United States over double dry matter per-capita per year (DM cap/yr.) is produced for animal feed than for foodstuffs (Krausmann et al., 2008). Livestock diet can therefore be exclusively forage or largely forage with concentrate supplementation. Concentrate supplementation is used to compensate nutritional deficiencies in the forage supply, increase animal performance such as milk production or at particularly challenging periods of development, for example calving. Due to most livestock diet being of forage this review focuses on the main crops used worldwide and will not discuss concentrates. The amount plant science has contributed to improvements in concentrates has been underappreciated and undervalued in literature, however, the role these crops have on livestock production has been reviewed previously (Erb et al., 2012). Forage crops can be grown in mixed species cultivation to provide nutritional and environmental benefits. By offering livestock mixed grazing pastures or blending feeds, nutritional quality can be enhanced. For example, alfalfa is the highest-yielding perennial forage legume and produces more protein per unit area than other forage legumes and so can be grown alone or in combination with a range of different grass species. Well-managed alfalfa is normally grown successively for 3 or more years, but if harvested too late in the season the crop cannot survive the winter (Bélanger et al., 2006). ### FORAGE NUTRITIONAL CONTENT ### Digestibility The nutritional status of a forage crop depends upon the concentration (and ratios) of carbohydrates, proteins, and lipids. The composition of these organic nutrients determines the digestibility (D-value) of each crop which along with mineral and vitamins provides the amount of energy which can be derived by the animal (ME measured in MJ/kg DM) (Osbourn, 1980). Such calculations are becoming increasingly prevalent when growers are deciding which crop to grow based and particularly dependent on if the animal is non-ruminant or ruminant. In forage crops 50–80% of DM is carbohydrate; if this percentage is too low then supplements of grains can be added. The primary types of carbohydrate are the insoluble structural saccharides cellulose and hemicellulose, or the storage forms such as starch and water-soluble polymers (e.g., fructans). These are degraded into simple sugars through cleavage of glycosidic bonds, either by the animal itself (non-ruminant and ruminants) or via microbial digestion and subsequent animal absorption (ruminants only). Different ratios of carbohydrates within the forage crop will have altered downstream digestibility for the animal, especially if the cell-wall structure constrains digestion by the microbial population or limits plant cell wall penetration (Weimer, 1996). Although lignin, a polyphenolic compound within forage, is not a carbohydrate, it has a dramatic impact on the digestibility of cellulose hemicellulose; lignin binds with structural carbohydrates and cell wall proteins and reduces nutrient availability. For forages increased lignin concentration in the growing crop will increase the percentage of indigestible DM. Of the major forage crops grown globally grasses, particularly Lolium perenne, have high digestibility due to high soluble sugar content alongside low lignin content (Ruckle et al., 2017). Animal digestion of simple carbohydrates produces monosaccharides which can be readily metabolized. In ruminants, only microbial digestion of structural carbohydrates produces simple sugars which are subsequently metabolized to pyruvate. Pyruvate is absorbed by the animal and is metabolized further into volatile fatty acids (VFAs) which are a major energy source, (Bergman, 1990). Ruminants absorb VFAs in their rumen, and the rate of this is dependent on the concentration of individual VFAs, rumen pH and the absorptive area in the ruminal lining. ### Protein Nitrogen (N) availability to animals is predominantly from forage proteins and are estimated using crude total protein Kjeldahl measurements. Protein is usually abundant in the major form of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), although relative amounts vary between species (Wallace et al., 1997). This is especially true when comparing content in grasses with herbaceous legumes, with red clover (Trifolium pratense), white clover (Trifolium repens) and lucerne (Medicago sativa) grown widely due to their high protein value (Ruckle et al., 2017). Again, lignin will severely affect the digestibility of protein. Some micronutrients like proanthocyanidins or condensed tannins also change the digestibility of protein, but they inhibit protein degradation through binding. This can be advantageous as rapid protein degradation is causative of bloat, however, too high a tannin content will mean protein passing through the digestive track is unabsorbed and therefore a loss in nutrition value (Lees, 1992; Piluzza et al., 2014). This means there is a balance between reduced bloat and animal productivity (Mueller-Harvey, 2006). All grasses contain little or no proanthocyanidins, whereas many legumes especially big trefoil (Lotus pedunculatus) and Sericea lespedeza (Lespedeza cuneata) can have levels as high as 18% DM (Barry and Manley, 1984; Mueller-Harvey, 2006). Other N-containing compounds can be found in forage such as nucleic acids, nitrate and ammonia (Wallace et al., 1997). ### Lipids Lipids in forage crops are mostly found as polyunsaturated fatty acids (PUFAs) in the range of 10 – 30 g kg−<sup>1</sup> (Hatfield et al., 2007) of which the most abundant is α-linolenic acid [62% total lipids (Clapham et al., 2005)], with linolenic and palmitic acid also being present (Harfoot and Hazlewood, 1988). These dietary lipids are important in final animal product quality; forage diets with lower PUFA levels than cereal diets can produce leaner meat (Wood et al., 2004; Van Elswyk and McNeill, 2014). Moreover, fresh forage has been shown through numerous studies to produce milk with lowered PUFA content and increased trans-fatty acids (Elgersma et al., 2006; Chilliard et al., 2007). Studies have been used to profile PUFAs across forage species, with grasses tending to have more α-linolenic acid when compared to legumes and legumes in turn having higher linolenic acid content (Boufaïed et al., 2003). Striking differences in PUFA content can be seen within species through profiling cultivars, and moreover the harvest period and its environment (Elgersma et al., 2003; Clapham et al., 2005). For example Lolium perenne, Festuca pratensis (meadow fescue), and Festulolium hybrids of the two have been shown to vary not only between species at the beginning of their growth season, but more prominently between individual cutting regimes (Dewhurst et al., 2001). ### Trace Elements Minerals and trace elements from forages are important for maintaining livestock health. As there is a move toward using fewer antibiotics in animal production the nutritional balance of feed takes on additional importance. Zinc is particularly important for the immune system and supplements can be added to animal feed, but addition of too much results in wasteful excretion, reviewed in Brugger and Windisch (2015). Contrastingly, avoiding accumulation of toxic minerals can also be important for forage crops. Getting the balance right is crucial as low levels of selenium can be beneficial for livestock, but high concentrations are toxic (Zhu et al., 2009). Some elements accumulated in plants can make them unpalatable for livestock, but the ability of forage crops to grow fast and quickly recover from cutting makes them ideal crops for phytoremediation [e.g., Napier grass, (Ishii et al., 2015)]. ### Biomass Production Probably the most important trait of any forage crop is rapid biomass production, as crops are either cut or grazed directly, and nutritional quality depends on the rate of biomass production. Intensive production with faster growth often decreases this nutritional, but this depends on the species grown and some cultivars have better recovery from defoliation. Plant height correlates well with biomass for most crops (e.g., maize) and this factor together with ground area cover are the criteria underpinning methods to assess yields (Freeman et al., 2007). Many plant species can be grown for forage production, but the ability of the shoot meristem to respond with increased growth after cutting is essential. In some forage species, aboveground grazing or cutting has been correlated with increased root exudation (Paterson and Sim, 1999). This flush of carbon release by roots can stimulate rhizosphere microbes that in turn help to mobilize soil nutrients to sustain aboveground regrowth. Maintaining an optimal nutrient and water supply is very important for forage biomass production. For example, the importance of N supply for re-growth after cutting grass has been demonstrated (Dawson et al., 2004). Furthermore, the previous N status of alfalfa influences its regrowth ability (Meuriot et al., 2004, 2005). ### IMPROVING FORAGE CROPS ### Cultivar Breeding Due to the relatively recent cultivation of forage crops compared to other agricultural plant species, there were few improvements before 1900. Recently, agricultural trends and the global economic importance of forages, mean new cultivars have been bred. These improvements are helped by many closely related wild populations which can be used in development of new lines (Boller and Green, 2010). The most desirable improvements are increasing dry matter yield (DMY), crop durability and resistance to diseases particularly by pathogenic fungus and pests particularly nematodes, digestibility of DM, and nutritional content of this tissue. Arguably the greatest improvements have been made in breeding of Medicago spp., Trifolium spp., Lolium, and Festuca. Large scale breeding programs include testing of these crops, such as NE1010, a multistate cooperative effort of 15 institutes across 12 North-eastern states of United States and Canada (NIMSS, 2017). Similar tropical grass breeding programs include the Brachiaria partnership between the International Centre for Tropical Agriculture based in Colombia (C.I.A.T.), the Ugandan National Livestock Resources Research Institute (NaLIRI), the Tanzania Livestock Research Institute (TALIRI), the Institute of Agricultural Research of Mozambique (IIAM) and the Brazilian Agricultural Research Corporation (E.M.B.R.A.P.A.) (CIAT and CGIAR, 2015) which is being conducted across Eastern and Southern Africa. Breeding programs for forage crops are fraught with difficulties. Individual plants have high genotypic and phenotypic heterogeneity with many species being polyploid, a problem which is exacerbated by in-breeding across many grasses, and few agronomic traits being linked to distinct genes (Poehlman, 1987; Vogel and Pedersen, 1993). Studies have focussed on this problem in specific legumes (Jahufer et al., 2002; Riday and Brummer, 2007; Collins et al., 2012; Luo et al., 2016) and grasses (de Araüjo et al., 2002; Piano et al., 2007; Blackmore et al., 2016). Regardless of these problems there have been some major developments in breeding lines for forages, especially in Medicago and Lolium. Figure 2* shows a brief historical timeline of Lolium cultivation, and includes the current breeding regimes for grasses; future breeding possibilities are also included and discussed in later sections. One of the most interesting breeding developments is the exploitation of closely related species of Lolium and Festuca (Thomas and Humphreys, 1991; Humphreys et al., 2003) to create hybrid Festulolium cultivars. These cultivars have the high quality characteristics of Lolium combined with the stress tolerance and persistence found in Festuca (Ghesquière et al., 2010). Backcrossing of Festulolium have generated novel hybrids with more stable protein content when compared to parental lines (Humphreys et al., 2014). Advances in phenotyping are making it easier to include the quantification of characteristics in the field; such as high level imaging of growing crops to accurately determine later traits like biomass (Walter et al., 2012). New cultivars are being helped by advances in sequencing methods that can provide more transcriptomic data (Barrett et al., 2009; Pfeifer et al., 2013; Yates et al., 2014), including the identification of SNPs which may be investigated to improve Lolium (Blackmore et al., 2016) and Trifolium (Nagy et al., 2013). Draft genomes for such crops and cultivars are becoming increasingly common (Byrne et al., 2015; De Vega et al., 2015; VanBuren et al., 2015) as well as more evidence that model species like Brachypodium can direct research (Brkljacic et al., 2011; Rancour et al., 2012). Such research is providing clues to candidate genes which could be used for nutritional enhancement. ### Candidate Genes for Nutritional Enhancement Identification of potential candidate genes is usually through quantitative trait loci (QTL) analysis or marker-assisted selection (MAS) provided from the above completed genomes. Those identified are studied in relation to biomass and growth traits; in M. sativa QTL has been used for lodging resistance and vigor (McCord et al., 2014), plant height and regrowth following harvests in association with MsaciB (Robins et al., 2007), candidate gene analysis for flowering and stem height through CONSTANS-LIKE (Herrmann et al., 2010) and biochemical markers of ROS resistance genes for drought tolerance correlated to DM (Maghsoodi et al., 2017). The expression of other ROS associated genes of the Iron-Superoxide Dismutase family (Myouga et al., 2008) have also been linked to increases in DM in both the legume M. sativa (McKersie et al., 2000) and grass Lolium cultivars (Warnke et al., 2002). In Lolium, transcriptomics showing differentially expressed genes between wild-type and a dwarf mutant enabled identification of three key genes associated with dwarfism (Li W. et al., 2017), which were subsequently used for forward screens. Markers are used to infer both phenotypic traits and to track inheritance to aid breeding. For instance chloroplast SSRs have been investigated in Lolium through a similar technique as above (Diekmann et al., 2012). A thorough re-annotation of the model forage M. truncatula genome has also identified hundreds of small, secreted peptides coded by both macronutrient-responsive and nodulation-responsive genes, which could aid reverse genetics for improving many forage crops, especially M. sativa (de Bang et al., 2017). Iterative mapping software such as BioMercator (Sosnowski et al., 2012) has been used in Lolium to perform meta-QTL analysis using readily available published data (Shinozuka et al., 2012), consequently providing new candidate genes from previous work including orthologs of rice amino acid biosynthesis genes and a marker for reproductive traits, showing how new algorithms can exploit old data. Moreover, BioMercator used to decipher flowering time and height in M. truncatula (Julier et al., 2007) directly implicated the above research into CONSTANS-LIKE in M. sativa (Herrmann et al., 2010; Julier et al., 2010). Such potential ease for transferring model plant knowledge to forage crop research is further discussed below. Despite the need to ensure optimal nutritional content especially in the end-product feed, rapid vegetative biomass accumulation is the most desirable trait of a good forage crop, especially those which undergo extensive cutting throughout the growing season. Due to this phenomenon, candidate genes for improving the crops are associated with either photosynthesis or nitrogen use efficiency (NUE). More generally for resistance to biotic and abiotic stresses there is also a huge opportunity for improving traits in forage crops using our genetic knowledge from model plants (see Figure 3). Figure 3 summarizes some of the traits that can be considered for all forage crops. Such improvements in traits could be aided by achievements in transformation and genetic marker techniques. Reproducible and high efficiency transformation has been developed for temperate cultivars of Festuca (Wang and Ge, 2005; Zhang et al., 2006) and Lolium (Bajaj et al., 2006; Badenhorst et al., 2016); and more recently for some of the tropical grasses such as Pennisetum (Gondo et al., 2017) and Brachiaria (Cabral et al., 2015). Some examples of gene editing forage crops to confer stress tolerances have been successful in aiding both biomass increases but also nutritional quality. Transformation of M. sativa with the Arabidopsis Enhanced Drought Tolerance1 gene produced plants with not only increases in root length, shoot height and vegetative biomass, but also increases in proline, soluble sugar and chlorophyll content under drought stress when compared to wild-type (Zheng et al., 2017). Importantly these increases were shown both in the laboratory but also in field conditions. This study also identified the increased expression of many interesting genes, including M. sativa Heat Shock Protein23 (HSP23), a gene already shown to enhance abiotic stress tolerance in both Nicotiana tabacum and Festuca (Lee et al., 2012a,b) along with other members of the MsHSP family (Li et al., 2016; Li Z. et al., 2017). Similarly, for the Ethylene Response Factor (ERF) family studies have been shown that introducing the M. sativa gene into other plants can confer enhanced resistance to salinity; MsERF9 and MsERF11 in Nicotiana and Arabidopsis, respectively (Chen et al., 2012a,b). ### Protein and N Budget Forage NUE is a target for breeding, particularly as protein content of crops is so valuable. Protein accumulation is linked to N status and when the supply is supra-optimal greater storage occurs. When compared with grain crops that have been bred for high seed starch, forage crops often require N in greater amounts due to their increased growth, storage capacity and higher fiber content (Parsons et al., 1991). For forage crops, it is the leaf tissue biomass that is harvested rather than grains/roots/tubers. Principally NUE for forage crops can be based on N utilization efficiency (NUtE) as we are interested in the highest achievable biomass of the shoot which will form the content to be dried for feed production (Xu et al., 2012). Not only does this include biomass, but also the relative N levels in this tissue; it is not enough to only have a high yield of biomass in the shoots, it must also yield optimal amounts of N. Moreover, when looking at the effect of fertilizer use we are also interested in how both the biomass and N status change on application and thus also N uptake efficiency (NUpE). Forage crops offer challenges for NUE as there is a requirement for optimal yield of shoot biomass with a high N content (NUtE) while also optimizing N fertilizer acquisition (NUpE) throughout the growth season. refinement method and bootstrapping = 100. The phylogeny was built using Newick format in iTOL v3.4.3 (Letunic and Bork, 2007) and a radial phylogenetic tree produced. The tree was color coded to show model species, human food crops and forage crops for both legumes and grasses, although it should be noted that many can overlap in their uses. As NUE is an important criterion for biomass improvements, many genes relating to N acquisition or metabolism have been the subject of study in model systems. Additionally, genes important in carbon metabolism have also been the focus, due to the links between C:N ratios for plant growth (Jaradat et al., 2009). Despite the long evolutionary divergence between grasses and legumes, many key candidate genes have high genetic similarity, meaning one can use known genes which effect a trait in a forage crop from one species and investigate it within another. For example, a range of vegetative N storage proteins have been identified and the reviewed for leaves (Muntz, 1998) and roots (Bewley, 2002). To illustrate this further, the phylogeny in Figure 4 is the known and predicted coding sequences for rbcS including the model species Arabidopsis thaliana, many significant grass and legume crops. Many of the forage crops have high similarity in their coding sequence to more well-studied crop species. For example, Medicago and Trifolium rbcS sit closely to the legume species which have their genomes sequenced [Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris (Jacob et al., 2016)]. Such sequences can provide a wealth of potential genes of interest for breeding programs (Araújo et al., 2015; Rauf et al., 2016). For example, investigation of Heat Shock Protein in M. truncatula, found a homologous HSP70 in M. sativa and had a substantial role in stress tolerance when conferred to A. thaliana (Li Z. et al., 2017). In forage crop vegetative biomass, the most important nutrients for livestock are proteins and water-soluble carbohydrates (WSCs), and ideally the post-harvest quality of these should be maintained. There has been considerable interest in developing organ specific proteome reference maps for stems and leaves. The dominant proteins in these tissues are photosynthetic enzymes such as RuBisCO and RuBiCO small unit (rbsS), which for M. truncatula make up ∼28.9% of leaf tissue, or other carbon-fixation genes for example glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase, with structural protein such as lignin biosynthesis being more concentrated in stems (Watson et al., 2003). As the D-value of forage is mostly linked to cell wall concentration and a reduction of this can aid digestibility (Jung and Allen, 1995; Jung et al., 2012), some proteomes have looked even more specifically at such tissues (Gokulakannan and Niehaus, 2010). Some research has focussed on transgenic approaches to increase and enhance amino acids and proteins. As many forages have low concentrations of the sulfur-containing amino acids of methionine and cysteine, both important in animal and human nutrition (Ball et al., 2006), some studies have specifically aimed at increasing these levels by over-expression. These have included using lupins (Lupinus albus) (Molvig et al., 1997; Tabe et al., 2010) and soybean (Dinkins et al., 2001; Tabe and Droux, 2002), used as forage sources. Apart from cultivar differences which can be improved with breeding programs or specific transgenic approaches, the most significant changes in nutritional content is due to stresses (Araújo et al., 2015). Consequently, stress proteomes have also been used for vegetative tissue; lupin stem proteins have been analyzed under water stress to show increases in serine protease and cysteine protease required for remobilization of proteins (Pinheiro et al., 2005); in grasspea (Lathyrus sativus) seedlings under either salinity, low temperature or ABA stress gave rise to the identification of 48 stress-responsive proteins (SRPs) which include those important dominant proteins discussed above (Chattopadhyay et al., 2011); in M. sativa drought conditions showed remobilization of RuBisCO-derived N could compensate for the decreases in N assimilation (Aranjuelo et al., 2011). Moreover, through harvesting regimes, forage crops undergo extreme stress which has shown to cause the remobilization of vegetative storage proteins (VSPs) to boost new shoot regrowth in both Medicago and Trifolium as well as being important for cultivars with better cold tolerance (Avice et al., 2003), whereas Lolium has shown how defoliation increases the relative proportions of certain proteins, particularly asparagine and glutamine (Bigot et al., 1991). Finally, the N consumed by livestock is recycled and increasing ruminant productivity is a major target for as the conversion of plant to microbial protein is inefficient. It was estimated that as much as 70% of the plant N eaten by animals for milk or meat production is excreted as ammonia or urea to the environment (MacRae and Ulyatt, 1974; Kingston-Smith et al., 2008; Kingston-Smith et al., 2010). Furthermore, the process of rumen fermentation is important for the generation of greenhouse gasses like methane (Bannink et al., 2008; Dijkstra et al., 2011). ### Rhizosphere Microbiome The impact of genomics extends beyond the crop plants to their environmental interface. For example, the rhizosphere microbiome is likely to be a future target for improving the nutritional quality of crops. Epiphytic bacteria living on and in the plant, may be important for crop health and nutrition, and some microorganisms can fix atmospheric N within legume root systems. Bacteria living with plants may be able to assist in digestion and absorption of forage eaten by livestock. These bacteria may improve the uptake of trace elements in the animal gut by the production of specific binding molecules and/or siderophores. In the soil, the rhizosphere microbiome is important for nutrient cycling and uptake, particularly in low input systems like those grown in the tropics. The inoculation of new forage crops with beneficial microorganisms is likely to be a target for research and use in future crops, coupled with rhizosphere microbiome research of root exudate composition. The root is known to directly modify the rhizosphere population by altering the chemical constituents of root exudates. For example, the roots of the tropical grass Brachiaria specifically produce a chemical shown to inhibit nitrifying bacteria and to specifically block ammonia-oxidizing pathways in soil bacteria, the first step in the process of converting ammonium to nitrate (Byrnes et al., 2017). Soil ammonia-oxidizing bacteria quickly convert urea or NH<sup>4</sup> <sup>+</sup> fertilizer to NO<sup>3</sup> <sup>−</sup>. Soil N form is fundamental for crop acquisition, as NO<sup>3</sup> <sup>−</sup> is mobile and readily leached while NH<sup>4</sup> <sup>+</sup> binds. Nitrification inhibitors have been identified in root exudates from several legumes and grasses including sorghum and rice, but by far the largest activity was detected in the tropical grass Brachiaria humidicola (Subbarao et al., 2009). In rice, the ability of root exudates to inhibit nitrification varied between cultivars from 5 to 50%, but was not significantly higher in three ancestral lines (Tanaka et al., 2010). The biological nitrification inhibitor (BNI) activity of root exudates has been assayed using a recombinant luminescent reporter ammonia-oxidizing bacteria Nitrosomonas europaea (Subbarao et al., 2006). In Brachiaria, roots exudate the cyclic diterpene "brachialactone," (Subbarao et al., 2009); brachialactone has a 5-8-5-membered ring system and a γ-lactone ring and contributed to 60–90% of the BNI activity released from the roots of this tropical grass. This exciting example offers the potential for transferring this trait to other forage crops to improve NUE. In the future synthetic pathways to produce plant nitrification inhibitors will be fully elucidated, providing the opportunity to capture this trait in forages and transfer to other crops to improve yield and nitrogen acquisition. ### Digestibility As protein digestion and uptake in livestock is directly related to energy availability (ME) (Nocek and Russell, 1988; McCarthy et al., 1989) it is important to increase WSC in many forage crops, especially grasses (Miller et al., 2001). In Lolium WSCs include fructans which are the most important storage polysaccharide and thus improved metabolism of fructan from sucrose can help improve the D-value (Chalmers et al., 2005). Use of distinct Fructan:Fructan 6G-fructosyltransferase sequence variants has shown to increase fructan levels at warmer temperatures in Lolium, thus hoping to aid development of high sugar-content grasses even at changing climates (Rasmussen et al., 2014). The amounts of WSCs are strongly associated with the N availability to the root (Roche et al., 2017) highlighting the importance of C:N balance in vegetative tissue (Louahlia et al., 2008). Furthermore, the amounts of WSCs varies between varieties as well as within the environment; Lolium cultivars AberMagic, AberDart, and AberElite all had highest growth rates correlated to highest WSC concentration during spring/summer, corresponding to high N availability from the roots alongside optimal photosynthesis conditions (Winters et al., 2010). Recent advances in the identification and manipulation of photosynthesis promoters for both Lolium perenne RBCS and Chlorophyll a/b Binding (CAB) (Panter et al., 2017) has provided transgenic lines for assessing increases in yield, fiber and, more importantly for digestibility, the fructan concentrations in both pseudostem and leaf blades in field trials (Badenhorst et al., 2018). Such work provides a platform for future studies to identify promotors important in other nutritional traits. The amounts of resistant starch are important for the digestibility and nutritional content of forage crops. Resistant starch (RS) generally has lower digestibility until it reaches the large intestine (Englyst et al., 1999), where in ruminants more digestion can occur (Raigond et al., 2015). Research studies have shown that M. sativa has advantages as a feed source over cereals for enhanced D-value (Giuberti et al., 2018). One major difference between dietary RS is that it is seen to have advantages in the human diet by providing more fiber, but disadvantages in livestock feed for non-ruminants as it remains undigested. In general, lower RS will improve the digestibility of forage crops for both ruminant and non-ruminant livestock. As a crops D-value is closely linked with its starch, protein and lignin content, genomic studies have begun large-scale genome-wide association studies (GWAS) to confirm correlations across a range of traits, such as using three distinct alfalfa cultivars with a high-throughput genotyping-by-sequencing approach (Biazzi et al., 2017). However, this study did highlight that differences in SNPs associated in different tissue types (shoots and leaves) can vary in correlation with traits such as protein content, and so care must be taken when using GWAS to aid crop improvements. Another substantial nutrient in forage crops is that of proanthocyanidins or condensed tannins (CTs). CTs bind to protein making it unavailable to digestion for ruminants until it reaches the rumen, and thusly an important trait in increasing the D-value of a crop (Min et al., 2003), although too high a CT content can be harmful restricting fermentation, especially in low leaf protein content species. A compromise is therefore desirable, with the moderate CT of 2–4% of the forage biomass giving the optimal D-value (Dixon et al., 2005). Whilst some species of legumes have optimum levels of CTs such as Lotus corniculatus, others such as Onobrychis viciifolia and Trifolium ambiguum are often poor choices for forage in many climates (Min et al., 2003; Baker, 2012); which means there is more scope to increase CTs concentrations in high yielding species where they are low such as M. sativa and Trifolium repens rather than increase growth traits aforementioned (Burggraaf et al., 2006; Salunkhe et al., 2017). As the CT synthesis pathway has been well-characterized in Arabidopsis with the transcriptional regulators R2R3 MYB, bHLH, and WD40 protein identified as having a central role in final CT content (Lepiniec et al., 2006). Such knowledge can be used to manipulate forage crops. The R2R3 MYB homolog MtPAR in the M. truncatula seed coat has been characterized and hairy root transformation in alfalfa resulted in the accumulation of CTs to the level of ∼20 mg/g shoot biomass (Verdier et al., 2012), although this is still below the desirable concentration. A similar study showed that expression of the TaMYB14 transcription factor from a low-yielding forage activates CT biosynthesis in both Trifolium and Medicago (Hancock et al., 2012). Other approaches have involved characterizing early steps of CT biosynthesis in M. truncatula in the hope to later target crop relatives (Pang et al., 2007), whilst others have looked at how relative amounts of CT differ between leaves and higher concentration containing flowers to see if changing flowering in Trifolium could improve its D-value (Burggraaf et al., 2008). There has been an effort to engineer better digestibility in some forage cultivars (Wang and Brummer, 2012) and microbial pre-digestion after cutting and before feeding, including microbial supplements (Boyd et al., 2011; West and Bernard, 2011; Elghandour et al., 2015), can be used to enhance this. ### Biomass Production As biomass yield is the main target for forage crop improvement, more rapidly growing cultivars can be targeted for breeding. Studies have consequently focussed on heading date (Fe et al., 2015) and flowering time regulation (Skøt et al., 2011; Shinozuka et al., 2012) in Lolium by developing Genomic Prediction models and QTL mapping as described previously (Nuñez and Yamada, 2017). Manipulating genes involved in delayed senescence has been targeted for increasing biomass yields. The introduction of the 5<sup>0</sup> flanking region of the Zea mays cysteine protease gene SEE1 in Lolium multiflorum has shown this promoter region to increase leaf lifespan by approximately 8–16 days (Li et al., 2004). A similar study using the Arabidopsis Senescence-Associated Gene12 (SAG12) promotor also delayed senescence in M. sativa with notable chlorophyll and yield increases even after 3 months of growth (Calderini et al., 2007). A final example is the expression of the Panicum virgatum NAC1 and NAC2 transcription factors in Arabidopsis atnap lines (mutants with defective senescence) to restore wild-type phenotype, predominantly measured using total chlorophyll concentrations (Yang et al., 2015). However, fast growth must also be coupled with the ability of the plants to respond to cutting by providing rapid regrowth. Growth rates and recovery from cutting are traits that are relatively easy to select for in breeding trials, and have been of regular interest to researchers for many decades in both Lolium and Medicago (Wilman et al., 1977; Vance et al., 1979). In addition, cutting experiments using 13C and 15N in both Lolium and Medicago have shown how the soil is affected both for dissolved organic C and N, and microbial biomass, demonstrating that management schemes can be critical to subsequent soil health (Schmitt et al., 2013). An ability to rapidly regrow may increase the susceptibility of the plant to insect and pathogens and this is worthy of further investigation. The relationship between tissue wounding and plant immunity is a topic that is quickly developing and there is now good evidence that tissue growth rate is closely linked with immunity (Huot et al., 2014). Thusly, management schemes for forage crops are very important for yield. For example, choosing when to cut or graze a crop is crucial for subsequent regrowth of the plant (Karn et al., 2006; Asaadi and Yazdi, 2011; Bumb et al., 2016). To assist in this choice there is scope for the use of molecular markers, with the future possibility of a PCR test for the optimal time harvest based on the expression of candidate genes like storage proteins. Such tissue testing of crops can also be used for decisions on the timing of fertilizer applications as the two evaluations are made at around the same time. There is scope to identify a suite of marker genes that can be used to help decide when these key decisions are made. Mixed cropping schemes are already widely used for forage crops and there are clear advantages in growing legumes and grasses together. Legumes increase soil N through their N fixation symbiosis with Rhizobium, with their biological nitrogen fixation ranging from 32 to 115 kg ha−<sup>1</sup> (Iannetta et al., 2016). This can in turn decrease subsequent fertilizer use for crops grown thereafter, a reduction between 23 and 31 kg N ha−<sup>1</sup> (Preissel et al., 2015). Numerous intercropping regimes have been tested including modeling of various climatic and soil texture parameters (Bachinger and Reining, 2009). Transfer of N from legume to crop, including in grasslands, has been investigated (Pirhofer-Walzl et al., 2012). However, it is still unknown how this interaction affects N movement and leaching through the soil profile. Such an investigation is required to give evidence of environment changes as well as crop productivity. Mixed species cultivation also has advantages for disease and extreme weather resistance as the susceptibility of the plants to these stresses varies between cultivars and species. Forage breeding has focussed on monoculture selection regimes and there is scope for better mixed species crops that could be included in trials for new varieties. Some advantages and disadvantages of mixed forage crops are summarized below in Table 1. Growing forage crops for improved nutritional quality has not been a target for breeding programs, rather yield and climate tolerance have been the drivers. Future crops must be tolerant of climate changes and weather extremes. Unlike many crops where monocropping is most productive, forage crops have the advantage that they can be easily grown in combination without lowering productivity. Such a trend has been shown across multiple trials as well as increasing biodiversity (Tilman et al., 2001, 2006; Weigelt et al., 2009). As with any system that promotes biodiversity whilst still being productive, this can TABLE 1 \| The advantages and disadvantages of growing forage crops in mixed systems. mean not only lowered costs to manage but also help cultural agriculture acceptability, with consumers becoming more aware of the effect the production of their food has environmentally (Scherr and McNeely, 2008). ### Trace Elements Plant research has focussed on the goal of biofortifying cereals, but there is also potential to improve the nutritional quality of forage crops. The economic importance of livestock production in the poorest parts of the world offers the opportunity to biofortify animal crops thereby improving the health of these animals and both directly and indirectly their owners. The knowledge base developed for grain biofortification (e.g., candidate plant metal transporters) has yet to be applied to forage crops. For example, transporter proteins for iron and zinc storage have been identified in cereals (Connorton et al., 2017; Menguer et al., 2017) and their equivalents in forages have yet to be identified. Although very abundant in most soils, silicon is particularly required by grasses (Tubana et al., 2016) and is therefore likely to be important for the optimal growth of many forage crops. Silicon is important for cell wall structure and therefore resistance to pathogens and pests, however, it may have a negative impact on digestibility. The supply of this nutrient may become limiting for forage crops, particularly as the plant biomass is regularly removed from the field and silicon is not yet a routine addition to fertilizer. Most species of forage crops can form mycorrhizal associations and this type of symbiosis is important for acquisition of trace elements. For natural grazing, these symbiotic associations are particularly important, but when fertilizer is added to cultivated forage crops mycorrhiza are suppressed (MacLean et al., 2017). Enhancing this symbiosis by inoculation of forage crops with mycorrhizal fungi has the potential to improve the mineral element composition of the feed. The fungal symbiosis has additional benefits for the plant by increasing the soil area mined for nutrients and water; this can be crucial during extreme weather events such as drought. Furthermore, a balanced and optimized root rhizosphere microbiome is essential for optimal root function and this applies to all crops including forage (Mommer et al., 2016). ### Environmental Footprint of Forage Crops As in all agriculture, improving water and nutrient use efficiency is a target for forage crops. The general fertilizer requirement of maize grown for forage and for grain are the same as that for a biomass crop. N requirements differ greatly for forage crops, and legumes and rhizome crops like Miscanthus have low N requirements (Dierking et al., 2017). Improving NUE using transporter marker genes as indicators of the crop status in the field could be valuable (Fan et al., 2017). Targeting particularly the NUpE component of NUE is important for minimizing the wasteful and environmentally damaging losses of excess N fertilizer additions. As discussed above for protein content, biomass production and cutting/grazing decisions there is the potential to develop gene markers that can indicate the N status of each type of forage crop. Mixed plant communities tend to have better NUE, probably because each species has a different temporal pattern of N uptake, resulting from different growth rates and root architecture (Tilman et al., 2001; Weigelt et al., 2009). In more affluent countries the relatively low chemical fertilizer prices do not encourage more judicious use of fertilizer for forage crops, but the threat of legislation for overuse has provided a new incentive for better fertilizer use efficiency. There is plenty of scope for improving the NUE of forage crops particularly as breeding programs have not focussed on this trail. For water acquisition, the long tap roots of Medicago are ideal for penetrating deep for water and nutrients. Varietal differences in this important trait have long been known (McIntosh and Miller, 1980) and the choice of cultivar depends on the soil type, climate and cropping regime that is required. ### CONCLUSIONS AND FUTURE DIRECTIONS ### Future Performance Improvements Using Genomics The availability of genomics and bioinformatics has revolutionized all biology and as databases expand to include more species and cultivars this information can assist forage breeders to improve crop performance. The future possibilities for breeding of forage crops using Lolium as an example are shown in Figure 2. By comparing cultivar sequence information and using GWAS for traits such as high vegetative tissue concentrations of protein, NUpE or specific trace elements the nutritional quality and yield of forage crops can be improved. Some SNPs in key genes that have been identified in model plants can be the targets for gene editing techniques (Bonhomme et al., 2014; Slavov et al., 2014; Thorogood et al., 2017). TILLING lines are also being used in many forage crops to study gene function (Carelli et al., 2013; Dalmais et al., 2013; Manzanares et al., 2016). Furthermore, as shown with the rbcS example in Figure 4, sequence information can be used for the design of PCR primers which can be used for tissue testing. These tests can be used to rapidly identify general health and nutritional status of crops as well as specific pathogens. One bottleneck is likely to be the transfer of the new genetic information into forage crops. For example, GM forage crops may be more acceptable to the public, as if fed to animals their entry into the human food chain is indirect. The use of CRISPR/Cas9 technology may provide an acceptable route for such manipulations, and as with many crops such feasibility studies have begun in forage crops; the mutation of the Medicago sativa Squamosa Promoter Binding Protein Like9 (SPL9) has been attempted and validated (Gao et al., 2018), although poor genome editing efficiency is limiting advances at present. Many candidate genes have been identified which may be quickly transferred into forage crops, but the technology for transformation is limiting development of these improved plants. In the future genome editing may become more accepted, particularly perhaps for animal feed crops. ### Focusing on Roots As discussed above high-yield, low-input vegetative biomass is desired for forage crop production. This has meant aboveground phenotyping strategies are being widely developed using predominantly imaging and spectral data (Walter et al., 2012), although more research is needed to see how vegetative phenotyping will work across different species, especially in mixed-cropping systems. However, although the need for welldeveloped, established root systems is clearly important (Kell, 2011; Nacry et al., 2013), breeding for belowground traits has been largely disregarded. This is unsurprising as with all crops, root phenotyping is difficult, being hidden in the soil and therefore labor intensive and difficult to sample. Any current root system improvements have been the consequence of vegetative drought and salinity assays discussed previously. Consequently, there has been a shift of focus toward breeding for underground traits in forage crops; across plant science this has been termed the next green revolution step (Lynch, 2007; Den Herder et al., 2010). Before phenotyping can even begin it is necessary to determine which kind of improvements are necessary, of which 2 main categories are found. The first is to improve root systems for the plant itself. This could include increasing fine root biomass, lateral root initiation, or in the case of legumes nodulation by Sinorhizobium, for increased nutrient uptake (Jackson et al., 1997; Ariel et al., 2010; Downie, 2010; Wang et al., 2010), or instead increasing root density or taproot length for either nutrient and/or water uptake, or resilience to stress such as defoliation (Dawson et al., 2004; Erice et al., 2007; Ghesquière et al., 2010; Kell, 2011). The second category is the improvement of root systems to aid the environment. This target is to improve agricultural land not just for production but also in terms of the ecosystem services, and this is especially true in the case of forage crops (Marshall et al., 2016). Forages and grasslands can provide ecosystem services that are wide-ranging and highly linked to root function including soil C-sequestration important for climate change (Kell, 2011, 2012), or lowering run-off of land thus helping to lessen flooding and soil erosion (Macleod et al., 2013). The idea of using both non-leguminous and leguminous forage crops as cover crops to mitigate climate change is gaining appreciation, (Kaye and Quemada, 2017). Another point to note is that many perennial grasses including Miscanthus and Panicum can be used for biofuel production but the characteristics required for a forage crop do not always match with those of a biofuel (Yang and Udvardi, 2018), although efficient root function and structure is likely to be a characteristic desirable for both agricultural sectors. Whether to improve plant performance or that of the environment, advances in phenotyping root systems will be crucial, including characterizing the plasticity of the system whilst the plant is growing. At present there are a plethora of root analysis software available (Paez-Garcia et al., 2015), but these require imaging roots either grown artificially such as on plates or already taken from the field and therefore evasive. There is therefore an increased interest in developing imaging techniques of plants grown in clear media to chart phenotypic changes throughout growth, or more promisingly the use of X-ray computed tomography (CT) scanning to give high resolution 3D models of the growing root system (Zhu et al., 2011). ### Developing Management Systems At present forage growers cannot easily and reliably determine the N status of their crops. For maximum biomass production, it is important to maintain the N status of the crop throughout the growing season and this requires an optimized soil N supply (Hofer et al., 2017). Application of too much N fertilizer results in wasteful run-off and sub-optimal supply results in decreased biomass production. Studies have already shown, through <sup>15</sup>N labeling of Lolium, how deficiency caused by low N fertilizer application causes an increase in the protein substrate pool whereas the store pool decreased in size and turnover rate (Lehmeier et al., 2013). This highlights the importance of fertilizer studies for N composition of forage crop vegetative tissue. Maintaining N supply for maximal yield is limited by two factors: (1) unreliable and unreproducible tests for soil N levels (Knight, 2006) and (2) an easy reliable measure of the crop's status. Presently farmers take limited samples across their growing area in the hope that this is representative of the N in the whole plot through the growing season. Nevertheless, this does not indicate a plant's N status or provide a measure of NUE. Some research has focused on the use of spectral data to evaluate crop efficiency (Foster et al., 2017), but such techniques require further investigation and can give false readings caused by pathogen attack. Sensors for N contents of soil are also being developed, however, these can be a costly solution (Shaw et al., 2016). Due to these problems, it may be better if the farmer could determine the crop N status directly and then make a more informed decision as to how they should subsequently fertilize the plot. This would enable more efficient fertilizer use, thus increasing forage biomass with lowered costs. Furthermore, for forage that includes legumes ### REFERENCES these N budget problems are complicated by the additional input of gaseous N-fixation. Other strategies of crop testing should be developed to reliably inform the grower of NUE efficiency. ### Final Animal Product Studies As forages are grown to rear livestock which in turn becomes food products for humans it is also important to view research in plant science from a livestock study prospective, of which has been touched upon above when discussing nutritional composition of crops. At present many countries adopt large-scale, concentratefeeding led livestock production like that of the United States, with many potential human health risks due to bacteria, antibiotic-resistant bacteria, prion, and dioxin presence in end products (Sapkota et al., 2007). Despite a rise in concentratefeeding, forage crops are still used widely as the main source of feed due to its high-yields of DM and energy for low costs (Reynolds, 2000), although usually studies focus on investigating a combination of both especially at various stages of development. For example, studies comparing growth of cattle fed a grass-diet instead of a linseed diet found the end product meat had a healthier fatty acid profile high in beneficial n-3 PUFAs, but the cattle were more slow-growing and thus the meat quality was poorer (Nuernberg et al., 2005). Similar outcomes have also been found for milk production from dairy cows in high-forage systems (Dewhurst et al., 2006). If improvements could be made in forage quality, especially more high-sugar varieties as outlined above, then potentially huge improvements in the animal production can be made. In conclusion, utilizing the information obtained from the research effort to improve grain crops and the knowledge gathered from model systems like Arabidopsis, offers an excellent future perspective for improving the nutritional quality and yield for forage crops. ### AUTHOR CONTRIBUTIONS NC and AM wrote the manuscript and conceived the perspective, read, and approved the final manuscript. ### FUNDING This project was supported by grants BB/J004588/1 and BB/J004561/1 from the Biotechnology and Biological Sciences Research Council (BBSRC) and the John Innes Foundation. NC was supported by an iCASE studentship from the BBSRC, grant BB/M015203/1, with the support of The British Association of Green Crop Driers Ltd. American Dietetic Association and Dietitians of Canada (2003). 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No use, distribution or reproduction is permitted which does not comply with these terms. # Joint Exploration of Favorable Haplotypes for Mineral Concentrations in Milled Grains of Rice (Oryza sativa L.) Guo-Min Zhang1†, Tian-Qing Zheng<sup>2</sup> \* † , Zhuo Chen3†, Yong-Li Wang<sup>1</sup> , Ying Wang<sup>1</sup> , Yu-Min Shi <sup>4</sup> , Chun-Chao Wang<sup>2</sup> , Li-Yan Zhang<sup>1</sup> , Jun-Tao Ma<sup>1</sup> , Ling-Wei Deng<sup>1</sup> , Wan Li <sup>1</sup> , Tian-Tian Xu<sup>2</sup> , Cheng-Zhi Liang<sup>3</sup> , Jian-Long Xu2,5 \* and Zhi-Kang Li 2,5 <sup>1</sup> Northern Japonica Rice Molecular Breeding Joint Research Center, Chinese Academy of Sciences, Haerbin, China, 2 Institute of Crop Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China, <sup>3</sup> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, <sup>4</sup> Guangxi Key Laboratory of Rice Genetics and Breeding, Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China, <sup>5</sup> Shenzhen Institute of Breeding for Innovation, Chinese Academy of Agricultural Sciences, Shenzhen, China ### Edited by: Huixia Shou, Zhejiang University, China ### Reviewed by: Yongzhong Xing, Huazhong Agricultural University, China Qingyao Shu, Zhejiang University, China \Correspondence: Tian-Qing Zheng [email protected] Jian-Long Xu [email protected]* †These authors have contributed equally to this work. #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 04 December 2017 Accepted: 22 March 2018 Published: 12 April 2018 ### Citation: Zhang G-M, Zheng T-Q, Chen Z, Wang Y-L, Wang Y, Shi Y-M, Wang C-C, Zhang L-Y, Ma J-T, Deng L-W, Li W, Xu T-T, Liang C-Z, Xu J-L and Li Z-K (2018) Joint Exploration of Favorable Haplotypes for Mineral Concentrations in Milled Grains of Rice (Oryza sativa L.). Front. Plant Sci. 9:447. doi: 10.3389/fpls.2018.00447 Grain minerals in rice, especially those in milled grains, are important sources of micro-nutrition elements, such as iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and selenium (Se), and of toxic heavy metal elements, especially cadmium (Cd), for populations consuming a rice diet. To date, the genetic mechanism underlying grain mineral concentrations (GMCs) in milled grain remains largely unknown. In this report, we adopted a set of 698 germplasms consisting of two subsets [indica/Xian (X-set) and japonica/Geng (G-set)], to detect quantitative trait loci (QTL) affecting GMC traits of Fe, Zn, Cd, Mn, Cu, and Se in milled grains. A total of 47 QTL regions, including 18 loci and 29 clusters (covering 62 Cd loci), responsible for the GMCs in milled grains were detected throughout the genome. A joint exploration of favorable haplotypes of candidate genes was carried out as follows: (1) By comparative mapping, 10 chromosome regions were found to be consistent with our previously detected QTL from linkage mapping. (2) Within eight of these regions on chromosomes 1, 4, 6, 7, and 8, candidate genes were identified in the genome annotation database. (3) A total of 192 candidate genes were then submitted to further haplotype analysis using million-scale single nucleotide polymorphisms (SNPs) from the X-set and the G-set. (4) Finally, 37 genes (19.3%) were found to be significant in the association between the QTL targeting traits and the haplotype variations by pair-wise comparison. (5) The phenotypic values for the haplotypes of each candidate were plotted. Three zinc finger (like) genes within two candidate QTL regions (qFe6-2 and qZn7), and three major GMC traits (Fe, Zn, and Cd) were picked as sample cases, in addition to non-exhausted cross validations, to elucidate this kind of association by trait value plotting. Taken together, our results, especially the 37 genes with favorable haplotype variations, will be useful for rice biofortification molecular breeding. Keywords: favorable haplotype joint exploration, grain mineral concentration, GMC, quantitative trait locus, QTL, milled grain, biofortification molecular breeding, rice (Oryza sativa L.) ### INTRODUCTION Micronutrient malnutrition (or "hidden hunger") is widespread throughout different countries (Kumssa et al., 2015), especially among poor populations whose daily caloric intake is confined to staple cereals (Gregorio and Htut, 2003; Ma et al., 2008; Bhullar and Gruissem, 2013). The development of biofortified cereals, especially mineral-dense rice, remains an efficient way to alleviate malnutrition in developing countries worldwide, including China (Gregorio and Htut, 2003; De Steur et al., 2012). Meanwhile, as a side effect of modernization, heavy metal pollution of arable land has become more and more severe; concentrations of toxic minerals, especially cadmium (Cd), are increasing in cereal grains, which threatens human health (Al-Saleh and Shinwari, 2001; Huang et al., 2007; Fu et al., 2008; Hang et al., 2009). Currently, with the fast expansion of rice cultivation to Northeast China, the grain mineral concentrations (GMCs) in early-matured japonica/Geng type rice have become more and more important in rice production. In addition to its relatively small genome, rice remains the world's most popular staple food crop (Dawe et al., 2010; GRiSP, 2013); therefore, both the biofortification and the relief of heavy metal pollution in rice have attracted increased research attention. The GMCs belong to complex traits controlled by multiple quantitative trait loci (QTL). Some QTL mapping studies have been carried out with different populations (Tang, 2007; Lu et al., 2008; Shen et al., 2008; Garcia-Oliveira et al., 2009; Zhang et al., 2009, 2011, 2014; Zhong, 2010; Anuradha et al., 2012; Bekele et al., 2013; Du et al., 2013; Kumar et al., 2014; Norton et al., 2014; Huang et al., 2015; Nawaz et al., 2015; Hu et al., 2016), and in-silico mapping (Chandel et al., 2011) for the GMCs in brown rice has been performed. GMC-related QTL tend gather in four regions on chromosomes 2, 3, 4, 6, 7, and 11, respectively. Specifically, there are three regions gathering QTL controlling Cd concentration in rice grains on chromosomes 4, 7, and 11, among which, the one on chromosome 7 is supported by evidence from four different tests. The single causative gene was identified as OsNramp1 (Ueno et al., 2009a,b, 2010; Ishikawa et al., 2010; Tezuka et al., 2010; Abe et al., 2011). However, just as the other cloned genes identified as associated with GMCs, such as, OsVIT (Zhang et al., 2012) and OsNAS (Lee et al., 2009) for Fe, OsLCT1 (Uraguchi et al., 2014) and OsHMA3 (Ueno et al., 2010) for Cd, OsNramp5 (Ishimaru et al., 2012; Liu et al., 2017; Tang et al., 2017) for Mn, and OsHMA4 (Huang et al., 2016) for Cu, it's also mainly responsible for the GMCs in the aleuronic layer rather than the endosperm, which is the major part of the milled grain. Currently, attempts have been made by a few molecular biologists using endosperm-specific promoters to improve the GMCs in milled grains (Zheng et al., 2010; Masuda et al., 2012). However, the genetic mechanism of GMCs in milled grains remains largely unknown. Previously, we used two sets of backcrossed inbred lines (BILs) derived from the same donor, and two elite new varieties in Southwestern China, Ce258 and Zhongguangxiang1 (ZGX1) as recipients, to assess the genetic background and the genotypic by environment (G × E) effects of GMC traits in rice milled grains using QTL mapping (Xu et al., 2015). Therefore, in the present study, QTL information from that linkage mapping work was used to confirm the results of a genome-wide association study (GWAS) using a set of 698 sequenced germplasms. Favorable haplotype joint exploration for candidate genes within important QTL regions was also carried out. ### MATERIALS AND METHODS ### Plant Materials and Field Experiments A set of 698 germplasms was adopted in this study. The set comprised two subsets, one was an indica/Xian subset (Xset) including 265 accessions randomly chosen from the 3K genome project (The 3,000 Rice genomes project, 2014), and the other was an early japonica/Geng subset (G-set), which included 433 accessions with sequencing data from similar sequencing pipelines. According to their maturation times, the X-set was planted at Sanya (18.3◦N, 109.3◦E) of Hainan province and the G-set was planted at Haerbin (45.8◦N, 126.65◦E) of Heilongjiang province. A small set of accessions was used as a control panel to check the variances between different environments. All of the above plant materials were transplanted into the field at a spacing of 13.2 cm between individuals and 25 cm between rows, with a final planting density of approximately 18,000 individuals per 667 m<sup>2</sup> . Field management was carried out according to the local farmers' practice. At the mature stage (about 40 days after flowering), seeds were bulk-harvested for each line. The seeds were air-dried and stored for 3 months in a drying house before being evaluated for the mineral concentrations (GMCs) in the milled grains. Basic physical and chemical properties of the soil in the paddy field were analyzed using routine analytical methods of agricultural chemistry (Lu, 1999). ### Evaluation of Grain Mineral Concentrations (GMCs) Dried seeds of each line were de-hulled, polished and then milled into flour, according to the surging and grind-milling method described in our previous report (Xu et al., 2015), to prevent possible mineral contamination, especially by Fe. About 0.3 g of rice flour was digested with 6 ml of HNO<sup>3</sup> and 0.2 ml of H2O<sup>2</sup> using a microwave digestion system (Microwave300, Anton PAAR, Graz, Austria), with the following parameters: 5 min at 700 W, 700–1,200 W for 10 min, and 1,200 W for 20 min. The samples were then transferred to a block heater at 160◦C for further digestion. The remaining 1 ml of digested sample was diluted with 50 ml of Milli-Q water before analysis. The Fe, Zn, Cd, Mn, Cu, and Se concentrations in the digested samples were determined using the methods described in our previous report (Xu et al., 2015). Two standards and two controls were set in each testing batch. Three replications of the tests were performed for each sample. ### Genotyping by Sequencing and Shared SNP Extraction The X-set germplasms were re-sequenced with an averaged depth of more than 10× (The 3,000 Rice genomes project, 2014). The cleaned reads were then mapped to the reference genome of Nipponbare (IRGSP1.0), and about 14 M high-quality single nucleotide polymorphisms (SNPs) were identified (The 3,000 Rice genomes project, 2014). Based on these 14 M SNP, a set of 2.9 M SNPs related to potential protein-coding areas was carefully selected. To build an SNP set for primary association studies, a subset of about 27,921 SNPs was selected from the 2.9 M SNPs by choosing one SNP per 100 counts, as described in our previous GWAS mapping work (Zhang et al., 2016). For the Gset germplasms, the quantity of the full set of SNPs was about 4 M. Finally, about 13 K SNP markers shared by both sets were extracted and submitted for further analyses, including sample clustering, principal component analysis (PCA), and GWAS mapping. These analyses were also carried out with the X-set and G-set data independently and compared with the pooled data. To perform deeper mining, favorable haplotypes were jointly explored for candidate genes within important QTL regions, based with the original 14 M and 4 M SNPs in the X-set and G-set, respectively. ### Data Analysis, QTL Mapping, and Haplotype Analysis Basic statistical analysis of the GMC traits, including the analysis of variance (ANOVA) and Duncan's t-test, were conducted using SAS software (S. A. S. I. Inc., 2004). The basic scenario of a compressed mixed linear model (Zhang et al., 2010), implemented in the Genomic Association and Prediction Integrated Tool (GAPIT) Version 2 (Lipka et al., 2012), was adopted for association analysis between QTL-flanking markers and GMC traits for the pooled, the X-set, and the G-set. Parameters for GAPIT were set with reference to our previous report (Zhang et al., 2016). A relatively stringent threshold was adopted to identify significant correlations between the SNPs and GMC traits, comprising a −log10(P)-value of 6.0. To minimize to the possibility of type II errors in QTL detection (Li, 2001), a relatively loose threshold of 3.0 was adopted for the loci with supporting evidence from our previous linkage mapping report (Xu et al., 2015) or other references. The allelic effects were estimated by setting the Major.allele.zero = TRUE in GAPIT Version 2 to identify the donors of favorable alleles and their effects on GMC traits. Subsequently, a joint exploration of favorable haplotypes was carried out according to the following steps: (1) By comparative mapping, we compared the results from the association mapping with the linkage mapping results from our previous report (Xu et al., 2015). The regions containing the jointly detected QTLs were then subjected to candidate gene analysis. (2) We searched the regions in the annotation dataset with wet-lab supporting evidence from the Rice Annotation Project database (RAP-DB) (Ohyanagi et al., 2006). (3) We then screened the genes by annotation information. If there were any obvious supporting evidence from the functional annotation, representing the relationships between the gene and the QTL targeting trait, then these genes would be highly focused in the next step. (4) Next, we compared all the mean values of the targeting traits for all the haplotypes of each candidate gene using pairwise comparisons with Duncan's t-test to identify significant associations between the variations of haplotypes and the QTL targeting traits. (5) Finally, we plotted the QTL targeting trait values for the haplotypes of each candidate in a straight-forward view. This joint haplotype exploration of the candidate genes was performed with the aid of Perl scripts and the full sets of SNPs in the X-set and G-set, respectively. For graphing and plotting, both Excel and R scripts were used. ## RESULTS ### Performance of the 698 Sequenced Accessions Among the 698 sequenced accessions, a wide range of variation was found for the GMC traits in the milled grains. As shown in Figures 1A–C, the concentrations of three major GMC traits (Fe, Zn, and Cd) ranged from 0.9 to 9.1 ppm, 5.8 to 29.6 ppm, and 0.002 to 0.054 ppm, with mean values of 2.4, 16.4, and 0.009 ppm, respectively. The concentrations of the other three GMC traits (Mn, Cu, and Se) (Figures 1D–F) ranged from 3.6 to 22.0 ppm, with a mean value of 9.7 ppm; from 0.8 to 7.5 ppm, with a mean value of 3.2 ppm; and from 0.01 to 0.11 ppm, with a mean value of 0.04, ppm, respectively. All the GMC traits fitted normal or normal-like distributions in the pooled set, except for the Cd concentration, which showed a binomial-like distribution (Figure 1). Notably, when we highlighted samples from the X-set and G-set with different colors, a major proportion of G-set samples were found to have higher Zn and Cu, but lower Cd concentrations. For the other three GMC traits, the phenotypic value distributions between the two sets overlapped markedly, especially for the Se concentration. The affects on the GMC trait values were caused by multiple factors, including different environmental conditions, especially the soil (Supplementary Table 1), as well as the genetic factors, were much more complex than we expected. Nevertheless, according to the ANOVA results based on the control panel (Supplementary Table 2), all the genotypic variances showed higher statistical significances than the environmental variances. Although limited by the diversity of the control panel, the effects of the genotypic variances for most GMC traits were only marginally significant or insignificant, except for the Zn and Mn concentrations. The Manhattan plots presenting the GWAS mapping results of the six GMC traits were shown in Figures 2A–F. Sample clustering and PCA analyses were also carried out based on the 13 K SNPs. The PCA result for the pooled data is shown in Figure 2G, and the kinship between the 698 accessions is presented in Figure 2H. For comparison, the PCA results obtained from the X-set and G-set independently are also shown in the Supplementary Figure 1. The results showed that the segregating pattern of the pooled set was quite similar to that of the X-set, whereas the G-set seemed relatively uniform. Considering that the optimum setting of the PCA value might vary according to different GMC traits, during the GWAS analysis with GAPIT, the Model.selection was set as TRUE for the optimum PCA value setting. (A–F) Distribution graphs for Fe, Zn, Cd, Mn, Cu, and Se concentrations in milled grains, respectively. ## Identification of Loci Controlling the Six GMC Traits According to the comparisons between the GWAS results from the pooled set and the two independent sets (X-set and G-set) shown in Supplementary Figures 2–7, a compensating mode was found between them. This meant that most signals in the pooled set were donated by either the X-set or the G-set, although the significance levels of the signals in the pooled set would be somewhat reduced if they were not significant in both subsets. To focus on the GMC QTL throughout different populations (also termed genetic background independent) and environments (also termed stably expressed), we adopted the results from the nucleotide polymorphism (SNP) genotyping data; (H) VanRaden map for the Kinship of the 698 germplasms. analyses based on the pooled set for further joint exploration of favorable haplotypes. A total of 47 QTL regions, including 18 loci and 29 clusters covering 62 Cd loci (Table 1, Figure 2) were detected by GWAS mapping for the six GMC traits from these 698 sequenced accessions. They included six loci for Fe, four loci for Zn, three loci for Mn, two loci for Cu, three loci for Se, and 62 loci belonging to 29 clusters for the Cd concentration. The average –log<sup>10</sup> value for these loci was 5.2 (range, 3.1–9.9). The –log<sup>10</sup> values varied by different GMC traits: It was 4.0 for Fe (range, 3.1–4.8), 3.5 for Zn (range, 3.4–3.6), 5.5 for Cd (range, 3.2–9.9), 4.3 for Mn (range, 4.0–4.6), 5.0 for Cu (range, 4.8–5.1), and 4.5 for Se (range, 3.8–5.6). Alleles from the germplasms increased the GMCs at about 38 (47.5%) of the above 80 loci, while they decreased the GMCs at the other 42 (52.5%) loci. Among the 42 loci with GMC decreasing alleles from the germplasms, 35 (83.3%) loci were responsible for the Cd concentration. However, among the 38 loci with GMC increasing alleles from the germplasms, 27 (71.1%) loci were responsible for the Cd concentration. Thus, according to the effects of GMC traits for human health, there were only 46 (57.5%) loci with favorable alleles from our 698 sequenced germplasms in comparison with the reference genome. According to their physical position, the 62 loci associated with the Cd concentration could be group into 29 QTL clusters TABLE 1 \| Quantitative trait loci (QTL) affecting grain mineral concentrations (GMCs) detected by a genome-wide association study (GWAS) in a panel of 698 germplasms. (Continued) #### TABLE 1 \| Continued <sup>a</sup>QTL cluster. <sup>b</sup>Favorable allele effect (FAE) values of the peak markers. <sup>c</sup>A GMCs-related QTL detected by linkage mapping in our previous report (Xu et al., 2015), in which the three parents for the BC populations were also involved in our germplasms for the GWAS mapping. <sup>d</sup>The number in brackets are reference codes as listed in reference section. (Table 1). Sixteen (55.2%) clusters harbored at least two loci (range, 2–5; mean = 3.6 loci/cluster). The three largest clusters were found on chromosomes 8, 11, and 12. Each of them harbored five loci for Cd concentration. Reverse allelic effects from the germplasms were detected for different loci gathered in one cluster. Among 14 (48.3%) of them, a single locus was found for each cluster. ### Haplotype Analysis of the GMC Candidate Regions We chose a total of 10 regions with supporting evidence from our linkage mapping for candidate gene scanning. A total of 192 coding genes with wet-lab evidence according to the RAP-DB (Ohyanagi et al., 2006) were identified in eight of the ten candidate regions (Supplementary Table 3). No significant relationship was found between the annotation information and the GMC traits; therefore, all 192 genes were submitted for further analysis. Candidate gene haplotype analysis was then carried out for these genes. Statistical comparisons between the mean values of the three major GMC traits (Fe, Zn, and Cd) were then carried out for different haplotypes of the genes in the X-set and G-set, respectively. Based on the results of Duncan'st-test for the haplotypes of the above candidate genes, 37 genes were found to have significant associations between the haplotype variations and the targeting trait of the QTL region (Table 2). There were no obvious GMC trait-related genes based on the annotation information from RAP-DB (Supplementary Table 3); therefore, three genes associated with zinc binding domain and/or zinc finger, which have not yet been reported to be related to the GMC traits, were chosen as sample cases in addition to non-exhausted cross validations. The genes were Os06g0489500 (Chr6:16404065- 17615233) for qFe6-2, and Os07g0568300 (Chr7:22841126- 22941126) and Os07g0569700 (Chr7:22841126-22941126) for qZn7. We performed trait value plotting for these samples following the above tests for all 192 candidate genes. We focused on the three major GMC traits: Fe, Zn, and Cd. Most phenotypic values between the different haplotypes for Os07g0569700 were insignificant, except for the Cd concentrations in the G-set. Thus, we only showed the significant results for the other two genes [Os06g0489500 (marginally associated with Fe) and Os07g0568300 (highly associated with Zn)] in Figures 3, 4 for the X-set and G-set data, respectively. From the X-set, among the five haplotypes of Os06g0489500 (Figures 3a,c,e), Hap3 seemed to be the most favorable one, which is associated with relatively higher Fe and Zn concentrations, but without significant affects on the Cd concentration, compared with the other haplotypes. Hap1 was the second choice. It was associated with an increased Fe concentration, but a relatively lower Zn concentration, and an insignificantly higher Cd concentration. Among the eight haplotypes of Os07g0568300 (Figures 3b,d,f), Hap6 seemed to be the most unfavorable one, being associated with relatively lower Fe and Zn concentrations, and an insignificantly higher Cd concentration. Hap4 and Hap8 from the X-set were only associated with higher Zn concentration and had no significant effect on the Fe or Cd concentrations. Additionally, mild but significant effects of Hap2, Hap5, and Hap7 on Zn concentrations were also detected compared with Hap6. In the G-set (Figure 4), among the nine haplotypes of Os06g0489500, Hap2 increased not only Zn but also Cd concentrations compared with the other haplotypes. Hap7 significantly reduced Zn, but had insignificantly increased the Cd concentration. Os07g0568300 was only associated with the Fe concentrations in the G-set. Among the seven haplotypes, Hap6 was favorable, which significantly increased the Fe, but had no significant effects on the Zn or Cd concentrations. In addition to these two significant candidate genes shown in sample cases, all 37 genes listed in Table 2 will become the focus for further functional verification in our future work. ### DISCUSSION ### Comparison of Identified GMC QTL With Reported Genes/QTL As described in another report for GMC QTL mapping in milled grain of rice (Hu et al., 2016), the statistical significances of QTL for the GMCs in milled grain are much lower compared with the QTL detected for GMCs in brown rice grains. This phenomenon also appeared in our association mapping experiment. Thus, we adopted two thresholds, including a relative loose one to minimize the type II error. Finally, we mapped a total of 80 loci (Table 1, Figure 2). Ten (12.5%) of them including qFe6-2, qFe7, qZn7, qZn12, qCd1-1/qCd1-2, qCd4-7, qCd6-2, qCd8-1, and qCd11-1 were consistent with the loci from our previous linkage mapping work, including qFe6, qFe7, qZn7, qZn12, qCd1, qCd4, qCd6, qCd8, and qCd11, respectively. Twenty (25%) of these 80 loci were also supported by loci reported in other works. Some were supported by multiple references. For example, qZn1 covered the region marked by id1005056–id1005058 (Norton et al., 2014), qZn7 was consistent with qZn7 (Huang et al., 2015; Hu et al., 2016) and qZN-7 (Lu et al., 2008), as well as the marker id7003641, which was significantly associated with the Zn concentration (Norton et al., 2014). QTL qCd7-1 was supported by qSCd7/ qGCd7 (Ishikawa et al., 2010) and qCdp7 (Abe et al., 2011). Some QTL were supported by single piece of evidence. The QTL qCd2-2 was consistent with qCd2b (Zhang et al., 2014). The loci qCd3-1, and qCd3-5 were consistent with two different reported qCd3 (Zhang et al., 2014; Huang et al., 2015), while the loci qCd11-8, and qCd11-9 were covered by a same relatively large region of qCd11 (Kashiwagi et al., 2009). The other 12 loci, qCd4-7, qCd5-1, qCd5- 7, qCd6-2, qCd6-3, qCd6-6, qCd7-2, qCd8-1, qCd8-3, qCd11-7, qCd12-9, and qCu5 were consistent with qCd4-2 (Kashiwagi et al., 2009), qCd5 (Zhang et al., 2014), qCd5.1 (Huang et al., 2015), Segment\_on\_Chr6 (Ishikawa et al., 2005), OsLCT1 (Uraguchi et al., 2014), qCd6 (Zhang et al., 2014), qCDCN-7 (Shen et al., 2008), Segment\_on\_Chr8(Ishikawa et al., 2005), qCd8 (Zhang et al., 2014), qCd11 (Tang, 2007), qSCd12 (Ishikawa et al., 2010), and qCu5 (Zhang et al., 2014), respectively. Notably, five loci (6.3%) including qZn7, qCd4-7, qCd6-2, and qCd8-1 were supported by multiple pieces of evidence from our linkage mapping and other references. Thus, they would be of higher value for breeding application, with characteristics of stable expression and/or genetic background independence. ### Multiple Evidence for QTL Detection for GMCs Although, the statistical significance for qFe6-2 and qZn7 was only marginal (both with –log<sup>10</sup> = 3.6) in GWAS mapping, they still possessed independent supporting evidence from the linkage mapping work (Xu et al., 2015). Additionally, there were supporting references of qZn7 (Huang et al., 2015; Hu et al., 2016), and qZN-7 (Lu et al., 2008), and the significant marker regions of id1005056–id1005058 (Norton et al., 2014). Thus, the joint application of the GWAS and linkage mapping again showed its power for QTL mapping, even for the traits with relatively low heritability, such as the GMC traits. Sometimes, multiple independent marginal evidences, when taken together, are more powerful than one single strong association signal. Japonica/Geng and indica/Xian differ markedly in their ability to accumulate Cd (Ueno et al., 2010; Uraguchi et al., 2011), which is much more significant than for the other GMC traits. Thus, when we pooled the two subsets together for the analysis, a population similar to those used for bulk-segregant analysis, with a bi-nominal distribution, was formed. This explained why the <sup>a</sup>QTL detected by linkage mapping in our previous report (Xu et al., 2015). <sup>b</sup>Loci detected by a genome-wide association study (GWAS) in this work. <sup>c</sup>The gene highlighted as sample cases in the latter part of haplotype analysis were shown in bold. <sup>d</sup>X-set = indica/Xian set, G-set = japonica / Geng set, \*, \\, \\\*, and \\\\ represents significant level of 0.05, 0.01, 0.001, and 0.0001, respectively, in the pair-wise comparison using Duncan's t-test for the different haplotypes of each gene. Cd QTL gained more statistical power in the GWAS mapping (Figures 1C, 2C). By contrast, the distributions of other GMC traits were not so significantly associated with the population structure, when divided by subsets (Figures 1A,B,D–F). In addition, only the locus significant in both sets, or at least highly significantly in one set, would be detected within the pooled data. Those peaks with an average level of significance in a single set would be highly likely to decrease in the analyses using the pooled data. However, this kind of underestimation of the QTL underlying the other five GMC traits would not have a large affect on the exploration of the really important loci that are suitable for practical breeding, especially those with multiple pieces of evidence that support the QTL, such as qZn7 and qFe6-2. Additionally, many closely linked QTL with reverse allelic effects for the Cd concentration were identified in QTL clusters along all the chromosomes, except for chromosomes 2 and 7. Thirteen (44.8%) of these clusters were supported by evidence from our previous mapping work or by other reports (Table 2). The largest clusters, Clst8C, Clst11b, and Clst12b, were each was found to harbor five loci for Cd concentration. Clst11b was also supported by evidence from multiple references. In our previous report, genetic overlaps were found for QTL controlling different GMC traits. Commonly, chromosomal crossovers in this kind of germplasm panel were thought to occur more frequently than in a bi-parental population. Thus, in this mapping work, with the improvement of mapping resolution compared with SSR linkage mapping in a bi-parental population, the details of the genetic overlap between GMC traits, especially those caused by tight linkage, may be magnified. The exact mechanisms underlying these Cd regions require further investigation. Finally, according to the joint favorable haplotype exploration, we found that functional annotation could not always offer sufficient useful information during the candidate genes screening. By contrast, the QTL targeting trait comparison would effectively help to narrow down the candidate genes from 192 to 37 by removing more than 80% unrelated information. ### Implications for Molecular Biofortification Breeding This work offers at least three useful implications for the biofortification molecular breeding of rice. The first is that the QTL or candidate gene haplotypes underlying the GMC traits detected in this report, as well as those from our previous report (Xu et al., 2015), showed multiple effects on more than one GMC trait. Thus, in biofortification molecular breeding work on crops, especially rice (Oryza sativa L.), a possible trade-off between the improvement of favorable GMCs, such as Fe and Zn, and the accumulation of toxic heavy metal elements, such as Cd, in the milled grain should be taken into consideration. Selection of favorable haplotypes of candidate genes during molecular breeding would decide the final success of the breeding products. For example, if we chose Hap3 from the X-set for Os06g0489500, a relatively higher Fe and Zn concentration in the milled grain would be obtained, together with an insignificantly lower Cd concentration; however, if Hap1 of the X-set was adopted, the improved Fe concentration would be accompanied by a relatively lower Zn concentration and an insignificantly higher Cd concentration (Figures 3a,c,e). Thus, when we construct a scheme for backcross (BC) breeding, which is commonly adopted in biofortification breeding, using certain germplasms with higher favorable GMCs, such as Fe and/or Zn, as donors and an elite line as recurrent parents (RPs), at least two important steps should be taken during parental selection. First, the existing haplotypes of the target genes in the RPs and donors should be clarified by genotyping and haplotype analysis. Second, different GMCs, especially nutrient minerals and toxic minerals, should be balanced. For different RPs, different elite donors with suitable haplotypes should be selected for crossing. The second point is that according to the mean values for the GMCs based on the haplotypes in the X-set and G-set, the Cd concentration is significantly lower in the G-set. This is consistent with known differences in Cd accumulation between indica/Xian and japonica/Geng (Ueno et al., 2010; Uraguchi et al., 2011). Thus, not only could the favorable haplotypes within the subspecies be used, but also those from across the subspecies could be taken into consideration. For example, for hybrid breeding, where most products belong to indica/Xian type, favorable haplotypes to decrease the unfavorable GMCs, such as Cd, could be imported from the japonica /Geng donors. Finally, by combining the joint exploration of the GWAS mapping results with the results from our previous linkage mapping work, and the reference data from other reports, it was possible to identify the QTL regions for the GMCs in the milled grain more reliably. All the mapped loci, especially those that were jointly detected, as well as their favorable haplotypes, offer an opportunity to enhance the Fe and/or Zn concentrations, but control Cd accumulation, in milled rice grains. Biofortification molecular breeding using the favorable haplotypes jointly explored in this work, involving marker assisted selection and/or gene editing, would be the next step of our on-going studies. ### AUTHOR CONTRIBUTIONS T-QZ, J-LX, and Z-KL: Conceived and designed the experiments; T-QZ, G-MZ, Y-MS, Y-LW, and YW: Performed the experiments; C-CW and T-QZ: Analyzed the data; ZC, C-ZL, T-TX, L-YZ, J-TM, L-WD, and WL: Contributed reagents, materials, and analysis tools; T-QZ and J-LX: Wrote the paper. ### REFERENCES ### FUNDING This work was supported by grants from the National Key R&D Program of China (2016YFD0101801), the Agricultural Science and Technology Innovation Program and the Cooperation and Innovation Mission (CAAS-XTCX2016009), the Open Funding Program from the Guangxi Key Laboratory of Rice Genetics and Breeding (160-380-16-3), the Scientific Program Guangxi Province (GuiKe AB16380119), the Chinese Academy of Sciences Strategic Priority Research Program Fund (XDA08020302), Helongjiang Province Science Fund for Distinguished Young Scholars (JC201214), the Shenzhen Peacock Plan, and the Green Super Rice Project Bill and Melinda Gates Foundation (OPPGD1393). ### ACKNOWLEDGMENTS We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00447/full#supplementary-material Supplementary Figure 1 \| Comparison of populations presented by clustering and principal component analysis (PCA) in three sets of data. Supplementary Figure 2 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Fe in the milled grains. Supplementary Figure 3 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Zn in the milled grains. Supplementary Figure 4 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Cd in the milled grains. Supplementary Figure 5 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Mn in the milled grains. Supplementary Figure 6 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Cu in the milled grains. Supplementary Figure 7 \| Comparison of genome-wide association study (GWAS) mapping results in three sets of data for Se in the milled grains. Supplementary Table 1 \| Physical and chemical characteristics of the soil in the experimental fields. Supplementary Table 2 \| Analysis of variance (ANOVA) for the control panel under two environments. Supplementary Table 3 \| Coding genes in the candidate regions used for haplotype analysis. accumulation in grain and aerial plant parts in rice. BMC Plant Biol. 9:8. doi: 10.1186/1471-2229-9-8 of Cd between roots and shoots in rice. Plant Cell Physiol. 50, 2223–2233. doi: 10.1093/pcp/pcp160 of brown rice grown in Cd-polluted soils. Euphytica 180, 173–179. doi: 10.1007/s10681-011-0346-9 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer QS and handling Editor declared their shared affiliation. Copyright © 2018 Zhang, Zheng, Chen, Wang, Wang, Shi, Wang, Zhang, Ma, Deng, Li, Xu, Liang, Xu and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Toward Eradication of B-Vitamin Deficiencies: Considerations for Crop Biofortification Simon Strobbe and Dominique Van Der Straeten\* Laboratory of Functional Plant Biology, Department of Biology, Ghent University, Ghent, Belgium 'Hidden hunger' involves insufficient intake of micronutrients and is estimated to affect over two billion people on a global scale. Malnutrition of vitamins and minerals is known to cause an alarming number of casualties, even in the developed world. Many staple crops, although serving as the main dietary component for large population groups, deliver inadequate amounts of micronutrients. Biofortification, the augmentation of natural micronutrient levels in crop products through breeding or genetic engineering, is a pivotal tool in the fight against micronutrient malnutrition (MNM). Although these approaches have shown to be successful in several species, a more extensive knowledge of plant metabolism and function of these micronutrients is required to refine and improve biofortification strategies. This review focuses on the relevant B-vitamins (B1, B6, and B9). First, the role of these vitamins in plant physiology is elaborated, as well their biosynthesis. Second, the rationale behind vitamin biofortification is illustrated in view of pathophysiology and epidemiology of the deficiency. Furthermore, advances in biofortification, via metabolic engineering or breeding, are presented. Finally, considerations on B-vitamin multi-biofortified crops are raised, comprising the possible interplay of these vitamins in planta. ### Edited by: Alexander Arthur Theodore Johnson, University of Melbourne, Australia ### Reviewed by: Francesco Di Gioia, University of Florida, United States Aymeric Goyer, Oregon State University, United States ### \Correspondence: Dominique Van Der Straeten [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 19 January 2018 Accepted: 21 March 2018 Published: 06 April 2018 #### Citation: Strobbe S and Van Der Straeten D (2018) Toward Eradication of B-Vitamin Deficiencies: Considerations for Crop Biofortification. Front. Plant Sci. 9:443. doi: 10.3389/fpls.2018.00443 Keywords: micronutrients, biofortification, metabolic engineering, folate, pyridoxine, thiamine, crop improvement, plant development ## INTRODUCTION In an era of tremendous technological capabilities, insufficient accessibility to nutritious food, a primary human need, still affects over two billion people on a global scale (Bailey et al., 2015; Gupta, 2017). Though approximately 800 million people endure energy deficit due to inadequate amounts of calories in their diet (Haddad et al., 2016; FAOSTAT, 2017), the relative abundance of undernourishment has dropped to almost half in the last 25 years. Unfortunately, the degree of undernourishment witnessed over the last decades has recently passed a minimum, as undernourishment is estimated to have affected 815 million people in 2016, as opposed to 777 million in 2015 (FAOSTAT, 2017). The general trend of a decrease in global undernourishment can be mainly attributed to yield improvement of important staple crops such as rice, maize and wheat, which has more than doubled since 1960 (Long et al., 2015). Unfortunately, caloric malnutrition represents only a portion of the food-related burden of diseases, as micronutrient malnutrition (MNM) is present in over one–fourth of the world's population (Bailey et al., 2015; Blancquaert et al., 2017; De Lepeleire et al., 2017). Micronutrient malnutrition comprises shortage of dietary micronutrients, including minerals (iron, zinc, iodine, selenium, etc.) as well as vitamins (Bailey et al., 2015). Micronutrient malnourishment, commonly referred to as the "hidden hunger," is known to induce diseases and disorders in many populations, not particularly confined to the developing world. Pregnant women and young children are most vulnerable for MNM, often resulting in death (Bailey et al., 2015; De Steur et al., 2015; Win, 2016; De Lepeleire et al., 2017). MNM can be considered an urgent global concern, persistent in many populations and remaining largely hidden (Ruel-Bergeron et al., 2015). Anemia, a condition of suboptimal hemoglobin level (Scott et al., 2014), illustrates the disastrous impact of combined micronutrient shortage on human physiology, as its occurrence has been linked with deficiency in iron (Camaschella, 2015), pro-vitamin A (Semba et al., 1992; West et al., 2007), thiamin (vitamin B1) (Franzese et al., 2017), pyridoxine (vitamin B6) (Clayton, 2006; Hisano et al., 2010) and folate (vitamin B9) (Moll and Davis, 2017). Anemia is held responsible for almost 2 million deaths of children under 5 years old on a yearly basis (Scott et al., 2014), and is estimated to affect more than 2 billion people globally. It is estimated that half of the cases of anemia could be attributed to deficiency in one or more micronutrients, though primary factors are iron and folate shortage (Scott et al., 2014). High incidence of micronutrient deficiencies are, in many cases, related to monotonous diets, largely consisting of energy rich, starchy staples (Blancquaert et al., 2017; De Lepeleire et al., 2017). These crops including wheat, rice, potato, cassava, corn and plantain have the tendency to contain inadequate levels of vitamins and therefore expose the population, consuming massive amounts of these staples, to the risk of vitamin deficiencies (Table 1). This is a downside of the cheap supply of energy rich staples, which enabled the aforementioned decrease in caloric malnourishment. Given the observation that MNM have a detrimental effect on global human health, there is a great need to strongly reduce these deficiencies, also stated in the Copenhagen consensus, where micronutrient interventions were ranked as the number one priority, related to Sustainable Development Goal 2 (SDG2), requiring great global investment (Copenhagen Consensus, 2012). Fortunately, there are several means to combat MNM in an effective way, which can be divided in education, supplementation and biofortification. Behavioral interventions, consisting of educational efforts encouraging dietary diversification, are the ideal means to improve the micronutrient status of a population (Reinbott et al., 2016). This strategy, however, requires changes in cultural or religious habits of certain communities, as well as recurrent interventions (Blancquaert et al., 2017). Fortification includes the administration of micronutrient to the population under the form of pills or fortification of food products (such as flour). The latter method, which is mandatory in many countries, has proven to be a rapid medium to ensure optimal micronutrient levels in the troubled populations (Pasricha et al., 2014; Sandjaja et al., 2015; Atta et al., 2016; Rautiainen et al., 2016; Wang et al., 2016). Unfortunately, supplementation depends on specialized infrastructure and appears difficult to implement in poor rural populations who have the highest demand for micronutrient interventions (Blancquaert et al., 2017). Luckily, biofortification, which involves the augmentation of the natural nutritional value of crops, can be addressed as a valuable additional strategy in the battle against MNM (Blancquaert et al., 2017; Garcia-Casal et al., 2017; Martin and Li, 2017; Strobbe and Van Der Straeten, 2017). Biofortification of locally consumed crops does not require changes in consumer behavior and demands only a one-time investment (De Steur et al., 2015, 2017; Bouis and Saltzman, 2017). Biofortification of staple crops, massively consumed in deficient populations, is an excellent way to supply sufficient micronutrients (Blancquaert et al., 2017; De Lepeleire et al., 2017). Biofortification sensu stricto, thereby omitting agricultural interventions (Cakmak and Kutman, 2017; Watanabe et al., 2017), comprises breeding techniques as well as genetic engineering approaches (Blancquaert et al., 2017; Bouis and Saltzman, 2017). Breeding strategies have the advantage to be easily implemented in agriculture, as they do not require exhaustive regulations (Mejia et al., 2017). However, the scope of the breeding approaches is confined to sexual compatibility, thereby lacking the ability to exploit useful animal or prokaryotic derived characteristics (Strobbe and Van Der Straeten, 2017). Biofortification via metabolic engineering, overrules this restriction. Furthermore, the latter approaches enable creation of a biofortification strategy blueprint, applicable to a wide variety of food crops (Strobbe and Van Der Straeten, 2017). Metabolic engineering does, however, require a great knowledge of the specific micronutrient metabolism and its importance in the physiology of the plant. This review reflects upon the acquired knowledge which enabled successful B-vitamin biofortification in food crops, bundling information on thiamin (B1), pyridoxine (B6), and folates (B9). This evaluation includes vitamin function in plant growth and development as well as importance in human pathophysiology, epidemiology and accomplishments in biofortification. Furthermore, in view of multi-biofortification, the simultaneous biofortification of multiple vitamins and minerals, possible synergistic or adverse effects of micronutrient combinations, are scrutinized. Multi-biofortification endeavors are the step-stone for future eradication of MNM. A list of abbreviations can be found in Supplementary Table S1. ### VITAMIN B1 – THIAMIN Thiamin is a water soluble B-vitamin consisting of a pyrimidine ring, linked to a thiazole moiety by a methylene bridge (Lonsdale, 2006) (Figure 1). Vitamin B1 consists of different itamer forms of thiamin, predominantly occurring as thiamin and its different phosphate esters thiamin pyrophosphate (TPP) and thiamin monophosphate (TMP). However, other forms of thiamin do exist, such as thiamin triphosphate, though their contribution to the total pool is rather marginal (Gangolf et al., 2010). In literature, thiamin(e) is sometimes confusingly used to describe the total pool of the different B1-vitamers, here simply referred to ### TABLE 1 \| Vitamin content of six major staple crops. Vitamin content data were retrieved from the USDA database (USDA, 2016). RDA data were retrieved from (Trumbo et al., 2001). Values represent vitamin content of 100 g fresh edible portion of each crop. Fold enhancement needed to reach the RDA of the corresponding vitamin upon consuming the highest average national serving, is indicated between brackets. Data on staple crop consumption are derived from FAOSTAT (FAOSTAT, 2017). <sup>1</sup>Highest recommended daily allowance (RDA). <sup>2</sup> In 2013. <sup>3</sup>Milled equivalent. FIGURE 1 \| Thiamin biosynthesis in plants. Synthesis of pyrimidine and thiazole moieties as well as their condensation occurs in plastids. Biosynthesis pathway is shown in blue, enzymes in black. Transport across membranes is proposed to be carrier-mediated (barrels), of which the identified mitochondrial TPP carrier is indicated (red barrel) (Frelin et al., 2012). The chemical structure of thiamin is depicted, of which the free hydroxyl group can be pyrophosphorylated by action of thiamin pyrophosphokinase (TPK). End product feed-back, performed by TPP on the THIC riboswitch, is depicted in red. Products: Gly, glycine; NAD+, nicotinamide adenine dinucleotide; SAM, S-adenosylmethionine; AIR, 5-aminoimidazole ribonucleotide; HET-P, 4-methyl-5-β-hydroxyethylthiazole phosphate; HMP-P, 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate; HMP-PP, HMP-pyrophosphate; TMP, thiamin monophosphate; TPP, thiamin pyrophosphate. Enzymes: THIC, HMP-P synthase; THI1, HET-P synthase; TH1, HMP-P kinase/TMP pyrophosphorylase; TPK, thiamin pyrophosphokinase; TH2, TMP phosphatase, PALE GREEN1. as B1. The bioactive vitamer is TPP, serving as cofactor in multiple enzymatic reactions. ### Biosynthesis Biosynthesis of TPP in plants takes place in plastids, cytosol and mitochondria (Figure 1). Although the TMP vitamer is synthesized in the chloroplast, subsequent enzymatic reactions in mitochondria and cytosol are required to finalize the de novo biosynthesis of TPP, the bioactive vitamer (Mimura et al., 2016; Goyer, 2017; Hsieh et al., 2017). Formation of TMP involves the synthesis of the thiazole and pyrimidine moieties, followed by their condensation, all of which occur in chloroplasts (Goyer, 2017). The thiazole moiety of the thiamin structure is created by the action of 4-methyl-5 β -hydroxyethylthiazole phosphate (HET-P) synthase (THI1), requiring NAD<sup>+</sup> and glycine as a substrate and yielding HET-P (Godoi et al., 2006). In this reaction the THI1 enzymes is consumed, as it supplies a sulfide from a cysteine residue, making THI1 a 'suicidal' enzyme. 4-amino-2-methyl-5 hydroxymethylpyrimidine phosphate (HMP-P) synthase (THIC) is an iron-sulfur cluster containing enzyme, catalyzing the formation of HMP-P, the pyrimidine intermediate of thiamin biosynthesis (Raschke et al., 2007; Kong et al., 2008; Goyer, 2017). This reaction requires 5-aminoimidazole ribonucleotide (AIR) (derived from purine metabolism) and S-adenosylmethionine (SAM) as substrates (Chatterjee et al., 2008). This is the key regulatory step of thiamin biosynthesis, as the promotor is under control of the circadian clock, and the terminator is feedback inhibited by the end product TPP (Bocobza et al., 2013). This terminator contains a riboswitch, rarely seen in eukaryotes (Wachter et al., 2007). The riboswitch acts by binding TPP in its pre-mRNA state, resulting in the formation of an unstable splice variant of the THIC gene, thereby causing a lowered THIC activity (Wachter et al., 2007; Bocobza et al., 2013). Next, the bifunctional enzyme harboring HMP-P kinase and TMP pyrophosphorylase activities (TH1), catalyzes the HMP-P phosphorylation and subsequent condensation of HMP-PP and HET-P to form TMP (Ajjawi et al., 2007b). TMP forms the end product of plastidial thiamin biosynthesis and is further processed by the PALE GREEN1/TH2 enzyme, after exiting the plastids (Mimura et al., 2016; Hsieh et al., 2017). The subcellular localization of TH2 action, being cytosolic, mitochondrial, or both, has been debated. First, the enzyme was discovered in the cytosolic fraction of Arabidopsis (Ito et al., 2011; Mimura et al., 2016). Later experimental evidence, utilizing translational fusions with green fluorescent protein (GFP), confirmed the cytosolic location of the TH2 enzyme, realized from a (preferred) native secondary translational initiation site (Mimura et al., 2016). This secondary translational initiation site yields a protein which lacks the functional N-terminal mitochondrial targeting peptide of TH2. Therefore, TH2 was considered to be predominantly residing in the cytosol though likely also present in mitochondria. More recently, TH2 was found to be almost exclusively localized in the mitochondria of th2/pale green1 rescued with a GFP-fused TH2, controlled by the cauliflower mosaic virus (CaMV) 35S promoter (Hsieh et al., 2017). These seemingly contradicting findings can be explained by the fact that a strong constitutive promoter (35S) preceding the full coding sequence of TH2, favors the first translational start site and incorporates the mitochondrial targeting peptide into the arising protein (Hsieh et al., 2017). However, these findings highlight the ability of sole mitochondrial TH2 activity to complement the th2/pale green1 mutant, indirectly hinting at the existence/necessity of a mitochondrial TMP importer as well as a thiamin exporter. Combining these results, it can be concluded that TMP dephosphorylation, executed by TH2, likely occurs primarily in the cytosol and to a lesser extent in mitochondria. However, it cannot be excluded that this subcellular localization of TH2 action might change in different conditions/tissues/species. The reaction mediated by TH2 yields thiamin. In turn, thiamin, is the substrate for thiamin pyrophosphokinase (TPK), producing the active vitamer TPP in the cytosol (Ajjawi et al., 2007a). TPP subsequently travels to the different subcellular locations via carriers. Two mitochondrial TPP carriers have been partially characterized in plants (Frelin et al., 2012). ### Role in Plant Physiology Vitamin B1, sometimes called the 'energy vitamin,' plays a crucial role in plant energy homeostasis, mostly by the role of TPP as a cofactor (Goyer, 2010). TPP is a cofactor for three enzymes which are central to the energy metabolism. First, the pyruvate dehydrogenase (PDH) complex, catalyzing pyruvate decarboxylation, yielding acetyl CoA and NADH, necessary for the tricarboxylic acid (TCA or Krebs) cycle and biosynthetic processes, respectively (Bocobza et al., 2013). In addition, in the tricarboxylic acid cycle, α-keto-glutarate dehydrogenase (2-oxoglutarate dehydrogenase E1 component, OGDH) functioning also requires TPP, further accentuating its critical role in central metabolism (Rapala-Kozik et al., 2012). Third, TPP is an essential cofactor of the transketolase (TK) enzyme, playing a key role in the Calvin cycle as well as the pentose phosphate pathway, which renders pentose sugars as well as NADPH to the cell (Goyer, 2010). Hence B1, in its form of TPP, controls a few key steps in central aerobic energy metabolism. In this regard, TPP has been suggested to influence the flux through these pathways, as it is required in their rate-limiting steps (Bocobza et al., 2013). Indeed, modestly increasing TPP concentration, through introduction of a non-functional riboswitch in Arabidopsis, induced enlarged activity of TPP-dependent enzymes (PDH, OGDH, and TK) (Bocobza et al., 2013). Moreover, these plants emitted larger amounts of CO2, suggesting overactive oxidative metabolism. This is further confirmed by the observation of depleted starch reserves at the beginning of the light period in high TPP lines (Bocobza et al., 2013). This presents a clear rationale behind the strict circadian regulation on B1 biosynthesis. As a consequence of its influence on central metabolism, metabolite composition, particularly that of amino acids, is severely altered in plants with aberrant B1 composition (Bocobza et al., 2013). B1 metabolism was shown to have a clear function in enabling plants to cope with biotic as well as abiotic stresses. Thiamin biosynthesis as well as B1 levels were observed to increase upon application of abiotic stresses such as high light, drought, salt and oxidative stress, conferring tolerance (Kaya et al., 2015; Yee et al., 2016). Remarkably, this B1-induced tolerance to oxidative stress was concomitant with decreased production of reactive oxygen species (ROS) (Tunc-Ozdemir et al., 2009). The exact molecular basis for this role of B1 in stress adaptation of plants remains partly unknown. Given the enhanced expression of TPP-dependent enzymes in plants exposed to drought stress, the influence of B1 on abiotic stress control seems to be via its end product TPP (Rapala-Kozik et al., 2012). On the other hand, the B1 biosynthesis enzyme THI1, responsible for plastidial thiazole biosynthesis, appears to be able to directly regulate stomatal closure (Li et al., 2016). The potential of B1 to enhance abiotic stress tolerance was, however, not observed in engineered Arabidopsis lines (Dong et al., 2015). Considering biotic stresses, B1 has been shown to confer systemic acquired resistance (SAR) (Ahn et al., 2007; Bahuguna et al., 2012; Boubakri et al., 2012). Thiamin-treated plants depicted enhanced ROS (hydrogen peroxide, produced upon up-regulation of superoxide dismutase) accumulation upon infection (Bahuguna et al., 2012), contrasting their role of decreasing ROS in abiotic stresses (Tunc-Ozdemir et al., 2009; Kaya et al., 2015). By doing so, thiamin provokes priming, a state in which the plant has the ability to react more rapidly upon infection (Conrath et al., 2006; Ahn et al., 2007). This priming effect, described for B1, was confirmed in high B1 engineered Arabidopsis (Dong et al., 2015), but not seen in rice (Dong et al., 2016). Moreover, thiamin treatment of plants induced higher accumulation of phenolic compounds, salicylic acid (SA) (through higher phenylalanine ammonia lyase activity) and nitrogen assimilation (via increased nitrate reductase activity) (Bahuguna et al., 2012). ### Pathophysiology and Epidemiology The central role of B1 in central (oxidative) metabolism in humans is reflected in its pathophysiology upon vitamin deficiency. TPP plays an indispensable role in energy metabolism as a cofactor in cleavage of α-keto acids (Adeva-Andany et al., 2017), as well as general oxidative metabolism, identical to its role in planta. While having lost the ability to synthesize thiamin during their evolution, humans possess the potential to interconvert the different thiamin phosphate-esters (Zhao et al., 2001; Banka et al., 2014). B1 was the first vitamin for which deficiency was characterized, as it was considered a "vital amine' (hence 'vitamine'), defined as a substance inducing the disease beriberi upon insufficient consumption (Lonsdale, 2006). Beriberi is a disease occurring upon severe B1 deficiency, divided in wet and dry beriberi, depending on whether it is manifested in the cardiovascular system or in the peripheral nervous system, respectively (Abdou and Hazell, 2015). B1 deficiency can cause heart problems, and even lead to heart failure (Roman-Campos and Cruz, 2014). Different symptoms, such as enlarged heart and increased venous pressure, have been reported. B1 deficiency has also been linked to Sudden Infant Death Syndrome (SIDS), due to brainstem malfunctioning related to hypo-oxidative metabolism (Lonsdale, 2015). An insufficient supply of the B1-vitamin can induce severe alternation of the nervous system, which leads to a disorder called Wernicke's encephalopathy (WE) (Jung et al., 2012). WE involves the arising of selective brain lesions, the first symptoms of which include confusion, apathy and impaired awareness, eventually ending in coma and death. B1 deficiency-induced disorders are in many cases easily reverted with thiamin application, and often witnessed in patients suffering from chronic alcoholism (Butterworth, 1993). The detrimental effect of B1-deficiency on brain functioning can be explained by the strong dependency of the brain on the oxidative metabolism (Butterworth, 1993; Gibson et al., 2005). One of the greatest risks of B1-deficiency, along with the lethal consequences of untreated WE, is the difficulty of diagnosis, leaving many illnesses untreated (Harper, 2006). Although cases of severe beriberi have become rare, outbreaks of B1 deficiency-induced beriberi have been reported on a global scale, causing many deaths, even upon sufficient access to healthcare (Luxemburger et al., 2003; Ahoua et al., 2007). Hence, in developing countries, B1-deficiency often is not linked to the observed casualties (Barennes et al., 2015). Moreover, infantile exposure to B1-deficiency was recently shown to have longterm effects on motor functions and balance of the child (Harel et al., 2017). Furthermore, elderly people have been shown to be highly susceptible to B1-deficiency, even in the developed world (Hoffman, 2016). Indeed, an investigation in New York state (United States) identified 14% of elderly as being B1-deficient (Lee et al., 2000). B1-deficiency is likely to be exacerbating Alzheimer's disease, and could therefore be considered a serious threat, definitely not confined to the developing world (Gibson et al., 2013). Good sources of vitamin B1 are, besides animal-derived products (meats, liver, eggs, and dairy products), beans and peas, nuts and whole grains (Lonsdale, 2006; USDA, 2016). Different massively consumed crops, such as rice, cassava, potato and plantain contain inadequate amounts of B1 (Table 1). In the case of rice, polishing, which removes the aleurone layer to avoid rancidification, eliminates many nutritionally valuable substances, including B1 (Goyer, 2017). This is illustrated by the original observation of B1-deficiency induced paralysis and death in fowls fed with polished rice, reversible by administration of the rice polishings (Lonsdale, 2006). Therefore, overconsumption of such staples in a monotonous diet, imposes a serious threat to human health. Furthermore, high carbohydrate intake increases the need of dietary B1, which is explained by its role in carbohydrate catabolism (Elmadfa et al., 2001). This emphasizes the need for increased B1 levels in these popular starchy crops. ### Biofortification Engineering of the thiamin biosynthesis pathway to augment thiamin content in plants has been attempted recently (Dong et al., 2015, 2016). The key step in thiamin -and therefore B1 engineering is the first committed step in plastidial pyrimidine biosynthesis, THIC (Raschke et al., 2007). Activity of the THIC enzyme seems to be a major determinant of B1 biosynthesis, as indicated by the oscillations of the corresponding mRNA transcript with TMP levels (Bocobza et al., 2013). Moreover, this gene harbors a TPP-binding riboswitch in its 3<sup>0</sup> UTR, which enables it to destabilize its mRNA upon high TPP prevalence (Wachter et al., 2007). This feedback mechanism, rather unique in eukaryotes, further highlights THIC as a regulatory point in B1 biosynthesis and therefore the THIC gene as an ideal candidate in metabolic engineering approaches (Pourcel et al., 2013). Indeed, eliminating this riboswitch, thereby removing the feedback inhibition on THIC, elevates thiamin level 1.6-fold in Arabidopsis (Bocobza et al., 2013). Enhancing the flux toward biosynthesis of the pyrimidine intermediate is likely insufficient for accumulation of B1. Indeed, feeding Arabidopsis seedlings with the intermediates pyrimidine and thiazole indicates that both are necessary to achieve higher levels of B1 (Pourcel et al., 2013). Combined overexpression of THIC and THI1, the plastidial thiazole biosynthetic enzyme (Godoi et al., 2006), further enhanced B1 levels of Arabidopsis over threefold compared to wild type (Dong et al., 2015). Similarly, implementation of this combined engineering strategy in rice resulted in B1 increase of 2.5-fold in leaves and 5-fold in unpolished grains (Dong et al., 2016). However, B1 levels remained barely affected in polished rice seeds. Future engineering strategies in B1-biofortification will tackle additional bottlenecks in B1-accumulation as well as applying engineering strategies to specific tissues (Goyer, 2017). Taken into account the detrimental effects of THIC-riboswitch elimination, resulting in chlorotic plants with enhanced carbohydrate oxidation (Bocobza et al., 2013), B1 biofortification should be approached with caution. Besides metabolic engineering, there are opportunities to enhance B1 content in crops via breeding techniques. Indeed, several (wild) potato varieties were identified which harbor over 2-fold difference in B1 content compared to popular agricultural potato cultivars (Goyer and Sweek, 2011). Similarly, up to 2.7-fold variation was found in different cassava accessions (Mangel et al., 2017). Previously, over 10-fold B1 variation has been measured in rice (Kennedy and Burlingame, 2003). Recently, a genome wide association study (GWAS) identified multiple quantitative trait loci (QTL), underlying B1 content in common wheat (Li et al., 2017). These results imply that breeding strategies could help in acquiring higher B1 levels in popular/regional crop varieties. On the other hand, elevating of B1 levels through exposure to certain biological stresses has been suggested, as this proves to augment B1 biosynthesis by significantly increasing the expression of the biosynthesis genes (Kamarudin et al., 2017). ### VITAMIN B6 Vitamin B6 represents a group of water-soluble molecules with similar biochemical properties, consisting of pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated esters (Fudge et al., 2017). PN, PL and PM differ by carrying a hydroxymethyl, an aldehyde or an aminomethyl substituent, respectively (Figure 2) (Hellmann and Mooney, 2010). Considering these six vitamers, the phosphorylated pyridoxal (PLP, Figure 2D) is the most bioactive, functioning as a cofactor in over a hundred reactions (Fudge et al., 2017). B6 can be considered a powerful antioxidant, comparable to carotenes (vitamin A) and tocopherols (vitamin E), as they are able to quench ROS (Bilski et al., 2000). pyridoxine-phosphate (PNP), (F) pyridoxamine-phosphate (PMP). ### Biosynthesis De novo biosynthesis of vitamin B6 takes place in the cytosol and comprises only two enzymes (Figure 3). Pyridoxal phosphate synthase protein (PDX1) generates pyridoxal 5<sup>0</sup> -phosphate (PLP) utilizing ammonia, glyceraldehyde 3-phosphate (G3P) and ribose 5<sup>0</sup> -phosphate (R5P) as substrates (Titiz et al., 2006). This ammonia originates from the reaction catalyzed by the PDX2 glutaminase, which releases ammonia from glutamine to yield glutamate (Tambasco-Studart et al., 2007). Furthermore, PMP/PNP oxidase (PDX3) is considered a crucial step in PLP salvage, ensuring its retrieval from the PMP and PNP vitamers (Sang et al., 2007). The non-phosphorylated vitamers PL, PM, and PN, can be converted to their corresponding phosphorylated vitamers by the action of the SALT OVERLY SENSITIVE 4 kinase (SOS4) (Shi et al., 2002). Finally, a pyridoxal reductase (PLR1) was identified, mediating a NADPH-requiring conversion of PL to PN (Herrero et al., 2011). Through these reactions plants are capable of balancing the different vitamer forms of B6, which is required to ensure controlled growth and development (Colinas et al., 2016). ### Role in Plant Physiology Vitamin B6 is involved in a plethora of metabolic reactions, serving as cofactor or required as an antioxidant (Tambasco-Studart et al., 2005; Mooney and Hellmann, 2010). PLP is considered to function as a cofactor for about 200 enzymatic reactions in Arabidopsis (Fudge et al., 2017). These PLPdependent enzymes, covering oxidoreductases, transferases, hydrolases, lyases, and isomerases, can be explored using the B6 database tool (Percudani and Peracchi, 2009). These reactions roughly cover the whole spectrum of plant metabolism. In doing so, B6 is required in amino acid synthesis as well as catabolism (Mooney and Hellmann, 2010). This is illustrated by the Arabidopsis mutant reduced sugar response (rsr4-1), harboring a mutated B6 biosynthesis gene (PDX1), exhibiting a decreased content of shikimate, altered levels of different amino acids, and higher levels of TCA constituents (malate, citrate and fumarate) (Wagner et al., 2006). Similarly, Arabidopsis mutants for the PLP salvage enzyme PDX3 (involved in B6 vitamer interconversions) contained aberrant amino acid profiles. The initial step in starch breakdown, α-glucan phosphorylase, requires PLP as a cofactor (Mooney and Hellmann, 2010). Furthermore, PLP-dependent enzymes play a role in synthesis of glucosinolates (Mikkelsen et al., 2004). Remarkably, biosynthesis of the plant hormones auxin (Zhao, 2010) and ethylene (Van de Poel and Van Der Straeten, 2014; Vanderstraeten and Van Der Straeten, 2017) as well as ethylene breakdown (Nascimento et al., 2014) involve PLP-requiring enzymes. B6 levels have also been linked to nitrogen metabolism as pdx3 lines were shown to be ammonium dependent (Colinas et al., 2016). This link is further strengthened by the observation that the ammonium transporter mutant amt1 has altered B6 levels (Pastor et al., 2014). On top of its vast influence on plant metabolism via PLPdepending enzymes, B6 plays a crucial role as an antioxidant (Vanderschuren et al., 2013; Fudge et al., 2017). Arabidopsis mutants with a lowered B6 status, exhibit distinct phenotypes including poor seed development, delayed flowering and reduced plant growth, while complete knock-outs are lethal (Vanderschuren et al., 2013). The lowered tolerance of these mutants to salt, high light, ultraviolet light, and oxidative stress illustrate the importance of B6 as a stress protector (Vanderschuren et al., 2013). Upon heat stress, a non-catalytic pyridoxine biosynthesis protein (PDX1.2), ensures sufficient B6 production by aiding its paralogs (PDX1.1 and PDX1.3), resulting in an increase of B6 content (Moccand et al., 2014; Dell'Aglio et al., 2017). Conversely, Arabidopsis lines, engineered for enhanced B6 content, display enhanced tolerance to abiotic stresses (Raschke et al., 2011). Furthermore, these plants exhibit enlarged cells, leading to larger organs. Interestingly, their amino acid and sugar composition is severely altered, reflecting the broad influence of B6 on plant metabolism. ### Pathophysiology and Epidemiology Vitamin B6, especially PLP, is crucial for correct human functioning, as it is required as a cofactor for around 4% of all enzyme activities (Ueland et al., 2017). Most of these reactions involve amino acid synthesis and catabolism, in which PLP serves as a cofactor in transaminations, aldol cleavages and carboxylations. Furthermore, PLP plays a role in energy metabolism as it is involved in gluconeogenesis and lipid metabolism. Moreover, B6 is necessary in the biosynthesis of heme as well as neurotransmitters (Ueland et al., 2017). In addition, B6 plays an important role as an antioxidant (Justiniano et al., 2017) and is even known to aid in enzyme folding (Cellini et al., 2014). In parallel with its functions in human metabolism, B6 deficiency is manifested in a broad spectrum of disorders. Most notably, B6 deficiency is known to provoke neurological disorders, such as peripheral neuropathy (Ghavanini and Kimpinski, 2014) and epileptic seizures (Skodda and Muller, 2013). Moreover, B6 deficiency might be linked to anemia, given the ability of B6 intake to cure some cases of the disease (Hisano et al., 2010). Furthermore, B6 deficiency has been associated with cardiovascular diseases, stroke, rheumatoid arthritis, diabetes and different types of cancer including colorectal, lung, breast, and kidney (Ueland et al., 2017). Although investigation on vitamin B6 deficiency on a global scale is lacking, there is evidence supporting the existence of persistent deficiency in several populations (Fudge et al., 2017). Indeed, studies in the United States and South Korea concluded that around one-in-four people have sub-optimal B6 status (Pfeiffer et al., 2013; Kim and Cho, 2014). Furthermore, half of the elderly in nursing homes in Norway were considered B6 deficient (Kjeldby et al., 2013). The situation in developing countries is estimated to be even worse, given the observation that over half of the population of Uganda and Sudan remain B6 deficient (Fudge et al., 2017). Knowing the detrimental effect this deficiency, remaining undiagnosed, could exert on human health, there is a strong need to supply these people with satisfactory amounts of B6. Humans, unable to synthesize B6 de novo, predominantly depend on their diet for sufficient B6 acquisition, as gut bacteria can be considered as suppliers of marginal amounts of different vitamins (LeBlanc et al., 2013; Fudge et al., 2017). Good sources of dietary B6, besides animal-derived products such as fish and meat, are fresh vegetables including carrots and onions (USDA, 2016; Fudge et al., 2017). However, bioavailability should be considered, given the observations that up to half of the B6 pool could be lost as a result of incomplete digestibility, which is found to be more problematic in plant-based food sources compared to animal products (Roth-Maier et al., 2002). Furthermore, the most consumed staple crops in the world are considered poor sources of dietary B6 (Fudge et al., 2017) (Table 1). ### Biofortification Metabolic engineering approaches rely on the knowledge acquired of the relatively simple plant B6 biosynthesis pathway, mainly involving PDX2 (Tambasco-Studart et al., 2007) and the pyridoxal phosphate synthase protein (PDX1) (Titiz et al., 2006). In a metabolic engineering strategy, overexpression of both PDX1 and PDX2 genes yielded up to fourfold increase in B6 levels, while overexpression of the single genes only generated marginal effects (Raschke et al., 2011). Interestingly, enhanced plant biomass in aerial organs with similar overall morphology as well as tolerance to oxidative stress were observed in two-gene engineered plants with increased B6 content. When targeted to roots, the two-gene approach, enabled almost sixfold augmentation of B6 in cassava, without any severe alteration in yield (Li et al., 2015). The success of this two-gene engineering strategy therefore supports assessment in different crops, as well as investigation of possible influences on crop physiology and yield. So far, analysis of crop germplasm has revealed limited variation (<2-fold) in B6 composition of potato (Mooney et al., 2013) and wheat (Shewry et al., 2011). However, screening of vast accessions of a particular crop could identify interesting lines and thereby also pinpoint novel important QTLs and maybe novel genes influencing B6 homeostasis (Fudge et al., 2017). ### VITAMIN B9 Folate is a collective term for a group of water soluble B9 vitamins. Folates can be considered tri-partite structures, consisting of a pterin ring linked to the para-aminobenzoate (p-ABA) moiety carrying a γ-linked glutamate tail (Scott et al., 2000; Rebeille et al., 2006) (Figure 4). The different folate species, called vitamers, are chemically different on three levels, being the oxidation state, the glutamate tail length and the nature of C1-substituents (Blancquaert et al., 2010; Strobbe and Van Der Straeten, 2017). These properties all exert an influence on folate stability. First, oxidized folates are considered more stable, given the susceptibility of the pterin – p-ABA linkage to (photo-) oxidative cleavage (Blancquaert et al., 2010). Tetrahydrofolates (THF), the most reduced folate forms, harboring a fully reduced B-ring in the pterin moiety, are the active cofactors. Conversely, folic acid, containing an aromatic pterin B-ring, is more stable, though exhibiting marginal natural occurrence (Blancquaert et al., 2010; Gorelova et al., 2017b). In this respect, the term 'folic acid' is used to indicate the synthetic folate analog. Second, folate entities greatly differ in their glutamate tail length, as they carry one to eight glutamates (Garratt et al., 2005; Strobbe and Van Der Straeten, 2017). Polyglutamylated folates are thought to possess enhanced in vivo stability as their polyglutamate tail ensures cellular retention as well as augmented association with folate dependent enzymes (Blancquaert et al., 2014). Third, folates species can differ in their attached C1- units, giving rise to an array of folate entities, affecting their stability and biological role (Figure 4). ### Biosynthesis In plants, folate biosynthesis is executed in different subcellular localizations (Figure 5). The pterin 'branch' resides in the cytosol (Strobbe and Van Der Straeten, 2017). Here, the first committed step is executed by GTP cyclohydrolase I (GTPCHI), utilizing GTP as a substrate and yielding 6-hydroxymethyldihydropterin (HMDHP) (Basset et al., 2002). An alleged pterin mitochondrial importer is considered to ensure translocation of HMDHP to the mitochondrion (Hanson and Gregory, 2011; Strobbe and Van Der Straeten, 2017). The plastidial p-ABA branch supplies the p-ABA moiety of the folate molecule (Figure 5). Here, the first committed step is performed by aminodeoxychorismate synthase (ADCS), using chorismate, originating from the shikimate pathway (Herrmann and Weaver, 1999), as a substrate (Sahr et al., 2006). Given the hydrophobic nature of p-ABA, it is thought to reach the mitochondria by diffusion through membranes (Hanson and Gregory, 2011; Strobbe and Van Der Straeten, 2017). Upon entering the mitochondria, HMDHP is pyrophosphorylated and coupled with p-ABA to form dihydropteroate. These enzymatic reactions are executed by the bifunctional HMDHP pyrophosphokinase/dihydropteroate synthase (HPPK/DHPS) (Gorelova et al., 2017a). Subsequently, dihydropteroate is converted to dihydrofolate (DHF) by the action of dihydrofolate synthetase (DHFS) (Ravanel et al., 2001), followed by a reduction catalyzed by dihydrofolate reductase as part of a bifunctional enzyme dihydrofolate reductase/thymidylate synthase (DHFR-TS) (Gorelova et al., 2017b), yielding THF. Folate biosynthesis is finalized upon polyglutamylation of THF, by the action of folylpolyglutamate synthetase (FPGS) (Ravanel et al., 2001; Mehrshahi et al., 2010). ### Role in Plant Physiology The chemical structure of folates makes them ideal carriers of C1-substituents, conferring a central role in carbon metabolism of nearly all living organisms (Blancquaert et al., 2010), with the exception of some Archaea (Gorelova et al., 2017b). Thereby, folates are both needed for proper anabolism as well as catabolism of cellular compounds. They play an essential role in the synthesis of purines as well as thymidylate and are therefore indispensable in DNA synthesis and growth (Stover, 2004). Furthermore, folates are required in biosynthesis of many plant metabolites including pantothenate (vitamin B5) and formyl methionyl tRNA as well as serine and glycine interconversion and catabolism of histidine (Blancquaert et al., 2010). Furthermore, folates are needed in production of lignin, ensuring cell wall rigidity (Srivastava et al., 2015). In addition, iron-sulfur cluster enzymes depend on folates for their assembly (Waller et al., 2010). Given their role as C1 donors and acceptors, folates play a key role in the methyl cycle (Blancquaert et al., 2010). 5-methyl-THF, is required as a methyl-donor in the conversion of homocysteine to methionine, which is necessary for replenishing of the SAM-pool (Blancquaert et al., 2010). SAM in its turn, functions as methyl storage in supplying this C1-unit to a wide range of methyltransferases, including DNA methyltransferases. Therefore, insufficient folate can alter the methyl-cycle homeostasis and evoke epigenetic changes by alteration in the DNA methylation pattern (Zhou et al., 2013). A disequilibrated folate homeostasis greatly influences epigenetic functioning through genome-wide hypomethylation, lowered histone methylation and transposon derepression, as witnessed in Arabidopsis methyleneTHF dehydrogenase/methenylTHF cyclohydrolase (MTHFD1) mutants (Groth et al., 2016). Similarly, aberrant functioning of FPGS, the enzyme responsible for extension of the glutamate tail, evoked upregulation of transposable elements (typically repressed by methylation), which could be reverted via administration of 5-methyl-THF (Zhou et al., 2013). Additional to their requirement in catabolism and anabolism of essential plant metabolites, folates appear to have a profound influence on plant growth and development. In non-photosynthetic plastids, the plastidial pool of folates influences plant energy metabolism by inhibiting starch formation (Hayashi et al., 2017). The mechanism is thought to operate via depletion of the ATP pool -required in starch assembly from sucrose- upon folate shortage, regulated by the folate-dependent DHFR-TS (Hayashi et al., 2017). Remarkably, the interplay of folate and sugar metabolism was shown to modulate auxin signaling, hence controlling plant development (Stokes et al., 2013). Moreover, folates possess the ability FIGURE 5 \| Folate biosynthesis is plants. Folate (vitamin B9) biosynthesis in plants occurs in three subcellular compartments: the cytosol, the plastids (green) and the mitochondrion (red). Biosynthesis pathway is shown in blue, enzymes in black. Polyglutamylated folates are considered the end product of folate biosynthesis. Products: ADC, aminodeoxychorismate; p-ABA, para-aminobenzoate; DHN-P3, dihydroneopterin triphosphate; DHN-P, dihydroneopterin monophosphate; DHN, dihydroneopterin; HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, HMDHP pyrophosphate; DHP, dihydropteroate; DHF, dihydrofolate; Glu, glutamate; THF, tetrahydrofolate. Enzymes: ADCS, ADC synthase; ADCL, ADC lyase; GTPCHI, GTP cyclohydrolase I; DHNTPPH, dihydroneopterin triphosphate pyrophophohydrolase; NSP, non-specific phosphatase; DHNA, DHN aldolase; HPPK, HMDHP pyrophosphokinase; DHPS, DHP synthase; DHFS, DHF synthetase; DHFR, DHF reductase; FPGS, folylpolyglutamate synthetase. to influence seed composition, demonstrated by the high N-content of Arabidopsis plastidial FPGS (atdfb-3) loss-offunction mutant seeds (Meng et al., 2014). This reveals an interaction between folate metabolism and N-metabolism in darkness. Folate metabolism was also shown to maintain root development in the indeterminate state, via FPGS functioning (Reyes-Hernandez et al., 2014). Folate synthesis and therefore accumulation is high during germination and in meristematic tissues, coherent with their demand upon cell division and concomitant DNA synthesis (Rebeille et al., 2006). Moreover, folate biosynthesis is stimulated upon light exposure, indicating a higher folate requirement (Rebeille et al., 2006). Indeed, the production of chlorophyll is dependent on folate (Van Wilder et al., 2009). Moreover, folates are able to ensure sufficient NADPH production, thereby controlling cellular redox state by a balanced functioning of DHFR-TS genes, needed in detoxification of ROS originating from photosynthesis or photorespiration (Gorelova et al., 2017b). In photorespiration, folate is directly required as a cofactor for the serine hydroxymethyltransferase in the glycine decarboxylase complex (Collakova et al., 2008; Maurino and Peterhansel, 2010). Finally, folate biosynthesis enzymes are known to influence plant stress responses, possibly through generation of folate biosynthesis intermediates (Storozhenko et al., 2007b; Navarrete et al., 2012). Given the influence of folates on plant development, their homeostasis and accumulation is considered to be tightly regulated, depending on their tissue specific requirement (Rebeille et al., 2006). Indeed, recent insights in folate metabolism of Arabidopsis confirm fine-tuning of folate accumulation by feed-back inhibition of a regulatory DHFR-TS homolog (DHFR-TS3) (Gorelova et al., 2017b). Together, these findings raise caution toward possible implications upon folate biofortification, as an increased folate pool might influence different aspects of plant physiology (Van Wilder et al., 2009). ### Pathophysiology and Epidemiology Humans lack the ability to synthetize folates de novo. However, they possess DHFR and FPGS enzymes, thereby allowing conversion of DHF to THF and polyglutamylated folates, respectively (Masters and Attardi, 1983; Garrow et al., 1992). Hence, humans are almost completely reliant on their diet for adequate folate supply, given that the gut microbiome has a marginal contribution to the folate pool (Camilo et al., 1996; LeBlanc et al., 2013). As the usage of folates as C1 donors and acceptors originated early in evolution, being implemented by prokaryotes and all eukaryotes, their basic functioning in plants is very similar to that in humans. Thus, folates are important in DNA synthesis and in supplying methyl groups to proteins, lipids, and DNA, through their necessity in SAM replenishment (Saini et al., 2016). Similar to plants, changes in folates levels have the potency to change the human epigenome (Bistulfi et al., 2010). Folates are required in methylation of myelin basic protein, which is pivotal for the compaction of myelin around the neuron sheath, thereby ensuring sufficient nerve conduction (Ramaekers and Blau, 2004; Bottiglieri, 2005). Upon inadequate dietary folate intake, folate status can drop, a condition known as folate deficiency, which has a broad pathophysiology. Folate deficiency results in decreased erythrocyte development, causing megaloblastic anemia (Lanzkowsky, 2016). The elevated levels of homocysteine, resulting from low folate status, can induce vascular diseases, such as coronary artery disease and strokes (Antoniades et al., 2009; Guo et al., 2009; Zeng et al., 2015). The most notable consequence of folate deficiency is its detrimental impact on neurulation. This is revealed by the occurrence of neural tube defects (NTDs) such as spina bifida, encephalocele and anencephaly, caused by folate deficiency (Geisel, 2003; Youngblood et al., 2013; Greene and Copp, 2014). Last but not least, different forms of cancer have been linked to inadequate folate status, including colorectal (Feng et al., 2017), prostate (Price et al., 2016), and pancreatic tumors (Yallew et al., 2017). Folate deficiency is still a global problem, predominantly present in the developing world, yet persisting in many populations of the developed world as well (Blancquaert et al., 2014; Zaganjor et al., 2015). Moreover, even populations blessed by the availability and opportunity of a diverse and folaterich diet, remain susceptible to deficiency, as illustrated by the low folate status measured in the Swedish population (Eussen et al., 2013; Gylling et al., 2014) and the observed sub-optimal folate levels in 39% of Belgian first trimester pregnancies (Vandevijvere et al., 2012). Worldwide, 300,000 pregnancies are estimated to be affected by NTDs annually, half of which are considered to be caused by insufficient maternal folate status (Flores et al., 2014). China, inhabited by almost 1.4 billion people, recorded a countrywide prevalence of NTDs as high as 0.24% (Blancquaert et al., 2014). More strikingly, Shanxi province, located in Northern China, has amongst the highest incidence rates of NTDs in the world, as high as 1.39% (Li et al., 2006). Fortunately, noteworthy advances have been made in the fight against folate malnutrition. Educational efforts, advocating a diverse diet containing folate rich foods such as green leafy vegetables and fermented products, is the primary strategy to diminish folate deficiency (Strobbe and Van Der Straeten, 2017). Folic acid, the synthetic form of folate as administered in pills, has been implemented in fortification strategies, which have ensured a significant reduction of neural tube defects (Williams et al., 2015; Wang et al., 2016). Unfortunately, high folic acid intake can also impose unwanted side effects, since excessive accumulation of unmetabolized folic acid has been linked to colorectal cancer and impaired immunity (Cho et al., 2015; Selhub and Rosenberg, 2016). Moreover, both folic acid fortification and supplementation are costly interventions, which are difficult to implement in poor rural regions in need (Blancquaert et al., 2014). Therefore, biofortification, via metabolic engineering or breeding is advised to ensure a stable cost-effective means to fight folate deficiency (De Steur et al., 2012, 2015; Blancquaert et al., 2014; Strobbe and Van Der Straeten, 2017). ### Biofortification Over the last decades, many successful folate biofortification approaches have been conducted, thereby additionally acquiring new insights in folate metabolism in certain crops and tissues (De Lepeleire et al., 2017; Strobbe and Van Der Straeten, 2017). The most widely attempted folate metabolic engineering approach is the enhancement of GTPCHI activity, proven to be a fruitful strategy in prokaryotes (Sybesma et al., 2003). This approach has been confirmed to be functional in plants by the engineering of cis-genic Arabidopsis lines, over-expressing GTPCHI (Hossain et al., 2004). This single gene approach, introducing GTPCHI, referred to as G-engineering, has been implemented in rice (Storozhenko et al., 2007a), tomato (de la Garza et al., 2004), maize (Naqvi et al., 2009), lettuce (Nunes et al., 2009), potato (Blancquaert et al., 2013a), and Mexican common bean (Ramírez Rivera et al., 2016). The highest fold enhancement, reached in the edible portions of these crops is a ninefold folate increase in lettuce. This could possibly be due to a difference in regulation in leafy tissue. However, single gene approaches have hitherto not resulted in over 10-fold increase in folate content. A bigenic approach was substantially more successful, adding ectopic expression of aminodeoxychorismate synthase (ADCS) (GA-strategy). In tomato (de la Garza et al., 2007) and rice (Storozhenko et al., 2007a) this led to 25- and 100-fold folate enhancement, respectively. Unfortunately, this approach, able to reach the desired levels in tomato and rice, does not promise to be universally applicable, as it only resulted in limited enhancements in Arabidopsis and potato (Blancquaert et al., 2013a). In rice seeds, ADCS has been indicated as the most important limiting factor in folate biosynthesis, additional to GTPCHI (Dong et al., 2014a). Building further on these findings, novel biofortification approaches aimed at further gene stacking, using mitochondrial folate biosynthesis genes (Strobbe and Van Der Straeten, 2017). Indeed, additional introduction of FPGS in GA-engineered plants did not only result in elevated folate levels in rice endosperm (100-fold) and potato tubers (12-fold) respectively, but also in enhanced folate stability upon storage (Blancquaert et al., 2015; De Lepeleire et al., 2017). Increasing storage stability has also been addressed by introduction of mammalian folate binding proteins (Blancquaert et al., 2015). This strategy is promising, as it could limit the aforementioned undesired effects of folate increase on plant physiology, via sequestration of the active folate pool. Moreover, recent discovery of plant folate binding proteins creates novel opportunities in folate biofortification via metabolic engineering (Puthusseri et al., 2018). Breeding endeavors, aimed at acquiring elite crop variants with augmented folate content in the edible portion, though not implemented so far, have shown to be feasible (Andersson et al., 2017; Bouis and Saltzman, 2017). Upon availability of high throughput folate quantification in the food matrix, screening of vast germplasm collections could lead to identification of high folate varieties (De Brouwer et al., 2010; Strobbe and Van Der Straeten, 2017). In this respect, over sevenfold variation in milled rice folate content was described by examination of 78 accessions (Dong et al., 2011). More recently, unpolished brown rice folate content was found to vary up to threefold in 150 examined accessions (Aiyswaraya et al., 2017). Similar screening has been employed in barley (Andersson et al., 2008), red beet (Wang and Goldman, 1996), potato (Goyer and Sweek, 2011; Robinson et al., 2015), tomato (Iniesta et al., 2009), muskmelon (Lester and Crosby, 2002), common bean (Khanal et al., 2011; Jha et al., 2015), lentil (Jha et al., 2015), (chick)pea (Jha et al., 2015), spinach (Shohag et al., 2011), and strawberry (Mezzetti et al., 2016). Furthermore, these variations could be utilized to identify interesting QTLs, underlying folate content, in GWAS (Khanal et al., 2011; Dong et al., 2014b). These techniques, though limited in their potential folate enhancement, are promising, as they might face lower regulatory restrictions, hence allow more rapid implementation in agriculture, reaching the populations in need (Mejia et al., 2017; Potrykus, 2017). ### B-VITAMIN INTERPLAY Multi-biofortification is considered an important goal in the fight against MNM (Blancquaert et al., 2014; Strobbe and Van Der Straeten, 2017). However, possible effects of altered micronutrient levels upon each other as well as on basic plant growth and development, should be taken into consideration. Examination of the role of B-vitamins in plant metabolism evidently reveals that inducing their accumulation could alter plant physiology. This has been conspicuously observed in metabolic engineering approaches of B1 (Bocobza et al., 2013; Dong et al., 2015) and B6 (Raschke et al., 2011). Furthermore, B9 enhancement, though not depicting any severe effect on plant growth, has shown to alter the rice seed metabolism (Blancquaert et al., 2013b). The influence of B-vitamins on plant metabolism is, however, at least partly intertwined, indicating the importance of detailed investigation of the effect of their combined biofortification (Figure 6). In central energy metabolism, both folate and B1 appear to negatively influence the plant's ability to accumulate starch (Bocobza et al., 2013; Hayashi et al., 2017). PLP (B6) is also involved in starch breakdown, though there are no indications to suspect increased starch breakdown upon elevation of PLP levels (Zeeman et al., 2004; Mooney and Hellmann, 2010). In the biosynthesis of B6, G3P, an intermediate in central energy metabolism (glycolysis), serves as a substrate (Fudge et al., 2017), the steady state concentration of which might be altered in B1 engineered lines (Bocobza et al., 2013). In altering this central metabolism equilibrium, B1 augmentation might influence the flux through the shikimate pathway (Bocobza et al., 2013), the activity of which is required in the plastidial part of folate biosynthesis (Strobbe and Van Der Straeten, 2017). In this shikimate pathway, PLP (B6) is required as cofactor. Folates are able to generate NADPH (Gorelova et al., 2017b), replenish the SAM pool (Blancquaert et al., 2010), and are needed in the biosynthesis of iron sulfur cluster enzymes (Waller et al., 2010). Interestingly, THIC, pinpointed as the rate limiting step in B1 biosynthesis, contains an iron-sulfur cluster and requires SAM for its catalytic activity (Pourcel et al., 2013). Strongly increased THIC activity would therefore require enhanced SAM turnover (Palmer and Downs, 2013), for which enhanced folate levels might have a beneficial effect. NADPH is on the other hand required for pyridoxal reductase activity in B6 homeostasis. Ribose 5<sup>0</sup> -phosphate, an important substrate in B6 biosynthesis (Fudge et al., 2017), is a product of the pentose phosphate pathway, the flux of which might be controlled by B1 (Bocobza et al., 2013). Similarly, AIR, an important substrate in B1 biosynthesis (Pourcel et al., 2013), is derived from purine metabolism, the synthesis of which is dependent on folate (Strobbe and Van Der Straeten, 2017). Moreover, B1, B6, and B9 have been linked to nitrogen metabolism. First, thiamin application is known to stimulate nitrogen assimilation (Bahuguna et al., 2012). Second, B6 content was observed to be altered in the ammonium transporter mutant amt1 (Pastor et al., 2014). Moreover, PLP (B6) salvage mutant pdx3 is depending on ammonium (Colinas et al., 2016). Third, folate biosynthesis mutants (atdfb-3, plastidial FPGS) harbored enhanced nitrogen content of seeds (Meng et al., 2014). Remarkably, given the labile nature of folate, increasing in planta stabilization of folates has been the subject of biofortification strategies (Blancquaert et al., 2015). Therefore, enhancing levels of antioxidants, such as B6, has been proposed as an additional biofortification strategy, protecting the folate pool from oxidative cleavage (Blancquaert et al., 2014). ### REFERENCES Abdou, E., and Hazell, A. S. (2015). Thiamine deficiency: an update of pathophysiologic mechanisms and future therapeutic ### FINAL REMARKS Creation and evaluation of multi-biofortified crops would not only offer a sustainable solution to eradicate MNM, but also help to elucidate the interplay of different micronutrients. The availability of novel tools, allowing facilitated cloning of multiple genes paved the way toward such multi-biofortification (Engler et al., 2014). Furthermore, a prerequisite in biofortification strategies is to consider stability upon storage of the crop product, as well as after food processing and bioavailability upon human consumption (Blancquaert et al., 2015; Diaz-Gomez et al., 2017). Different agronomical techniques could be employed, alone or in combination, to augment vitamin content of crops. Metabolic engineering of the complete pathway, or symbiosis with bacteria, might be appropriate ways to tackle vitamin B12 deficiency (DeMell and Holland, 2016). Metabolic engineering strategies could be developed in a precise way, enabling the creation of food crops which harbor an ideal balance of energy supply and micronutrient delivery, while exhibiting marginal effects on plant physiology. These novel crop varieties could, in combination with fortification and dietary interventions eradicate MNM, alleviating a great global burden. ### AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. ### FUNDING SS is indebted to the Agency for Innovation by Science and Technology in Flanders (IWT) for a predoctoral fellowship. DVDS acknowledges support from Ghent University (Bijzonder Onderzoeksfonds, BOF2009/G0A/004), and the Research Foundation—Flanders (FWO, project 3G012609). ### ACKNOWLEDGMENTS The authors thank Jolien De Lepeleire for the helpful suggestions and critical comments on the manuscript. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00443/ full#supplementary-material TABLE S1 \| An overview of the abbreviations. considerations. Neurochem. 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U.S.A. 107, 10412–10417. doi: 10.1073/pnas.0911586107 Conflict of Interest Statement:* The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Strobbe and Van Der Straeten. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # You Shall Not Pass: Root Vacuoles as a Symplastic Checkpoint for Metal Translocation to Shoots and Possible Application to Grain Nutritional Quality Felipe K. Ricachenevsky1,2 \, Artur T. de Araújo Junior<sup>2</sup> , Janette P. Fett2,3 and Raul A. Sperotto<sup>4</sup> <sup>1</sup> Departamento de Biologia, Programa de Pós-Graduação em Agrobiologia, Universidade Federal de Santa Maria, Santa Maria, Brazil, <sup>2</sup> Programa de Pós-Graduação em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, <sup>3</sup> Departamento de Botânica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, <sup>4</sup> Centro de Ciências Biológicas e da Saúde, Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari – UNIVATES, Lajeado, Brazil ### Edited by: Manuel González-Guerrero, Universidad Politécnica de Madrid (UPM), Spain ### Reviewed by: Seçkin Eroglu, ˘ ˙ Izmir University of Economics, Turkey Gian Pietro Di Sansebastiano, University of Salento, Italy > \Correspondence: Felipe K. Ricachenevsky [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 08 February 2018 Accepted: 14 March 2018 Published: 03 April 2018 ### Citation: Ricachenevsky FK, de Araújo Junior AT, Fett JP and Sperotto RA (2018) You Shall Not Pass: Root Vacuoles as a Symplastic Checkpoint for Metal Translocation to Shoots and Possible Application to Grain Nutritional Quality. Front. Plant Sci. 9:412. doi: 10.3389/fpls.2018.00412 Plant nutrient uptake is performed mostly by roots, which have to acquire nutrients while avoiding excessive amounts of essential and toxic elements. Apoplastic barriers such as the casparian strip and suberin deposition block free diffusion from the rhizosphere into the xylem, making selective plasma membrane transporters able to control elemental influx into the root symplast, efflux into the xylem and therefore shoot translocation. Additionally, transporters localized to the tonoplast of root cells have been demonstrated to regulate the shoot ionome, and may be important for seed elemental translocation. Here we review the role of vacuolar transporters in the detoxification of elements such as zinc (Zn), manganese (Mn), cadmium (Cd), cobalt (Co) and nickel (Ni) that are cotransported with iron (Fe) during the Fe deficiency response in Arabidopsis thaliana, and the possible conservation of this mechanism in rice (Oryza sativa). We also discuss the evidence that vacuolar transporters are linked to natural variation in shoot ionome in Arabidopsis and rice, indicating that vacuolar storage might be amenable to genetic engineering without strong phenotypical changes. Finally, we discuss the possible use of root's vacuolar transporters to increase the nutritional quality of crop grains. Keywords: biofortification, nutritional quality, vacuolar transport, metals, ionome ### ROOT APOPLASTIC AND SYMPLASTIC CONTROL OF METAL UPTAKE Roots are the primary sites of nutrient absorption and as such they must carefully control elemental uptake. This is accomplished via selective transporters at the plasma membrane of root cells at the epidermal and cortical cell layers. Root cells have their cytoplasm connected by plasmodesmata, membrane-lined channels that cross cell walls and allow diffusion of solutes between adjacent cells. The continuum cytoplasm-plasmodesmata of several cells make up the symplast (Rutschow et al., 2011). Once a molecule crosses an epidermal or cortical cell plasma membrane, it can move radially from the external layers into the internal stele and reach the pericycle by diffusion. Ricachenevsky et al. Root Vacuolar Transporters and the Ionome The next step is xylem loading, which is also dependent on membrane selective transporters that efflux nutrients out of the symplast. Thus, the symplastic route depends on the coordination between influx transporters at the external root cell layers, efflux transporters at the internal cell layers, and diffusion between cells that are symplastically connected (Miwa and Fujiwara, 2010). Indeed, influx and efflux transport systems characterized to date were shown to be important for the control of elemental concentrations in the xylem sap and consequently in the shoots (Figure 1A; Miwa and Fujiwara, 2010; Sasaki et al., 2016). Solutes from the rhizosphere can also move radially into root tissues penetrating extracellular spaces between cells and in cell walls, comprising the apoplast. However, at the endodermal cells, a lignin band deposited in anticlinal cell walls, the Casparian Strip, provides an extracellular barrier to prevent free diffusion into the stele through the apoplast (Figure 1A; Naseer et al., 2012). This cell wall modification interrupts the apoplastic communication from the external cell layers into the stele, thus making selective nutrient transport into the symplast necessary for ions to reach the xylem. Since endodermal cells contact both the external (connected to the rhizosphere) and internal (connected to the xylem) apoplast compartments, they are crucial to root nutrient uptake (Geldner, 2013; Barberon et al., 2016). The composition of the Casparian Strip bands and the mechanisms and genes involved in its formation during endodermal development are being dissected in detail in the model species Arabidopsis thaliana (Geldner, 2013). It was shown that Casparian Strips are actually made of lignin, not suberin (Naseer et al., 2012). Recently, it was demonstrated that suberin deposition on endodermal cell surfaces in response to nutritional stress could block access of apoplast solutes to plasma membranes, therefore making absorption by the epidermis and cortical cells necessary (Barberon et al., 2016). Thus, it is expected that changes in Casparian Strip bands and suberin lamellae deposition would result in altered radial nutrient movement in roots and modified access to the xylem and shoot translocation. Indeed, changes in Casparian Strip porosity results in leakage of nutrients from one apoplastic compartment to another, which changes xylem sap concentrations and consequently perturbs the shoot ionome (Hosmani et al., 2013; Kamiya et al., 2015; Huang and Salt, 2016). Thus, maintenance of diffusional barriers in the root apoplast is important for controlling root-to-shoot translocation of nutrients. ### ROOT CELL VACUOLES AS CHECKPOINTS FOR METAL DIFFUSION IN THE SYMPLAST AND ROOT-TO-SHOOT TRANSLOCATION IN ARABIDOPSIS: THE Fe DEFICIENCY EXAMPLE With proper apoplast diffusional barriers and the consequent symplastic control of absorption, the rate of uptake from FIGURE 1 \| Root vacuolar compartmentalization regulating the ionome. (A) Checkpoints of ion radial movement within roots. (1) Endodermal diffusion barriers block ions entry in the apoplast connected to the xylem. (2) Influx and (3) efflux transporters control ion concentration in the root symplast. Influx and efflux transporters may also be present in plasma membranes of other cells, but are shown in epidermis and pericycle for clarity. (4) Root vacuoles can restrict symplastic movement of ions, and therefore decrease or increase their availability for xylem loading and shoot/seed translocation. Different cell layers may have distinct vacuolar repertoire for storage. Gray arrows indicate diffusion within the symplast through plasmodesmata. Epi, epidermis; Cor, cortex; En, endodermis; Per, pericycle. Red band – Casparian Strip; yellow = suberin deposition. (B) Data from Genevestigator showing regulation under Fe deficiency of Arabidopsis genes AtIRT1 (AT4G19690), AtHMA3 (AT4G30120), AtFPN2 (At5G03570), AtMTP3 (AT3G58810), and AtMTP8 (AT3G58060). (C) Graphical visualization of the coexpressed gene network of the same genes as in (B) using ATTED-II (http://atted.jp/). Nodes (hexagons and circles) represent genes, while straight lines represent coexpression. Nodes in hexagonal shape and orange color represent genes of interest. Nodes in circle shape and purple color represent genes within the network which are directly connected to the genes of interest. Nodes in circle shape and light blue color represent genes which are connected to the purple circle genes. (D) Data from Genevestigator showing regulation under Fe deficiency of rice genes LOC\_Os03g46470 (OsIRT1), LOC\_Os02g43410 (OsYSL15), LOC\_Os07g12900 (OsHMA3), LOC\_Os05g03780 (OsMTP1), LOC\_Os03g12530 (OsMTP8.1), LOC\_Os02g53490 (OsMTP8.2), and LOC\_Os06g36450 (not yet characterized, but most similar gene to AtFPN1/AtFPN2; named OsFPN1). the soil and xylem loading presumably determines the concentration of a given element and the amount of rootto-shoot translocation. However, root vacuoles also control nutrients and trace elements concentrations in the root symplast (Figure 1A). Studies in Arabidopsis have shown that specific vacuolar transporters expressed in roots perform vacuolar compartmentalization, which can impact xylem loading and root-to-shoot translocation. Loss-of-function of these transporters result in higher translocation of respective elements to shoots, presumably due to increased element availability in the root symplast for efflux into the xylem (Arrivault et al., 2006; Morrissey et al., 2009). A striking example where vacuolar compartmentalization for multiple elements is part of a coordinated response, in which vacuoles detoxify elements that increase their concentrations due to excessive uptake, is observed during Fe deficiency response in Arabidopsis (Figures 1B,C). The classical Fe acquisition mechanism (reduction strategy, or strategy I) includes rhizosphere acidification by an H+-ATPase, Fe3<sup>+</sup> reduction to Fe2<sup>+</sup> by a membrane-bound, extracellular-facing reductase protein, and Fe2<sup>+</sup> uptake by the high affinity transporter AtIRT1 (Brumbarova et al., 2015). AtIRT1 has broad specificity, being able to transport other divalent metals, such as Zn2+, Mn2+, Co2+, Cd2+, and Ni2<sup>+</sup> (Korshunova et al., 1999; Barberon et al., 2014), which are potentially harmful. Indeed, increased concentrations of Zn, Mn, Co, and Cd in Arabidopsis shoots are part of the ionomics profile associated with physiologically Fe deficient plants, even if Fe concentration is not affected (Baxter et al., 2008). Recent work showed that non-Fe metals regulate AtIRT1 localization at the plasma membrane, which suggests that plants must balance Fe and other metal uptake through AtIRT1 under low Fe for optimal nutrition (Barberon et al., 2014). This observation indicates that AtIRT1 is the main route of entry for these metals, which transiently accumulate in roots of Fe deficient plants. The vacuolar transporters AtMTP3, AtMTP8, AtFPN2, and AtHMA3, which are, respectively, Zn, Mn, Co/Ni, and Cd/Zn transporters (Arrivault et al., 2006; Schaaf et al., 2006; Morel et al., 2009; Morrissey et al., 2009; Eroglu et al., 2016), are coordinately up regulated upon Fe deficiency, presumably in order to decrease local high concentrations in the root symplast (Figures 1B,C; Buckhout et al., 2009; Thomine and Vert, 2013). Consequently, their activity can reduce metal accumulation in shoot tissues. Therefore, the action of vacuolar transporters in compartmentalization of metals into root vacuoles indirectly control the shoot ionome, indicating that root vacuoles are a checkpoint for metal movement into the xylem and can fine-tune the accumulation of essential but/or potentially toxic elements in shoots. Interestingly, both AtFPN2 and AtHMA3 were shown to be involved in natural variation of Co and Cd shoot concentrations, respectively (Morrissey et al., 2009; Chao et al., 2012). Accessions harboring an insertion in the coding sequence of AtFPN2, which results in a truncated version of the protein, were hypersensitive to Co and Ni, and had increased concentrations of Co in shoots. The increased Co accumulation was more pronounced in conditions of low Fe availability, indicating that AtIRT1 uptake and AtFPN2 vacuolar compartmentalization work in concert to control Co movement in the symplastic xylem loading and root-to-shoot translocation (Morrissey et al., 2009). Regarding AtHMA3, a non-functional allele is present in several accessions of Arabidopsis, resulting in higher Cd concentration in shoots (Chao et al., 2012). AtHMA3 allele variation was considered the primary determinant of Cd concentration variation in shoots of Arabidopsis multiple accessions (Chao et al., 2012). These data suggest that vacuolar sequestration in roots might be important not only to general metal detoxification, but that fine tuning of detoxification could be involved in local adaptation of distinct genotypes within a species. Therefore, allele diversity of root vacuolar transporters might help to explain natural variation in the shoot ionome. ### ROOT VACUOLAR COMPARTMENTALIZATION UNDER Fe DEFICIENCY IN RICE There is little evidence for a conserved mechanism during Fe deficiency response in other species than Arabidopsis, although some of the orthologous genes are also up-regulated by Fe deficiency (Figure 1D). In rice, the best model species for monocots, Fe deficiency induces the combined strategy (with elements from both classical strategies I and II), which upregulates OsIRT1 (Ricachenevsky and Sperotto, 2014). However, evidence that OsIRT1 has broad specificity is still lacking, although over-expression of OsIRT1 leads to increased Fe, Zn, and Cd concentrations in rice plants (Lee and An, 2009). In rice, the MTP group 1 clade has only one member, named OsMTP1 (Ricachenevsky et al., 2013). OsMTP1 has been suggested to detoxify Zn into vacuoles as part of basal Zn tolerance, resembling the AtMTP1 function (Menguer et al., 2013; Ricachenevsky et al., 2015). Thus, an AtMTP3-like gene (i.e., with a function to detoxify high Zn under Fe deficiency) might be lacking in rice. Still, OsMTP1 may be somewhat involved in the Fe deficiency response, since expression data indicates it might be up-regulated in rice roots under low Fe conditions (Figure 1D). The rice ortholog of AtHMA3, named OsHMA3, has a role in Cd vacuolar detoxification in roots. Positional cloning and natural accession screening has shown that OsHMA3 is the causative gene of variation in Cd shoot concentrations (Miyadate et al., 2011; Yan et al., 2016). Conversely, overexpression of OsHMA3 resulted in increased Cd tolerance, with Cd concentrations increasing in roots and decreasing in shoots (Sasaki et al., 2014). These results show that OsHMA3 performs a similar role as AtHMA3, and that both are targets for variation in Cd concentrations within each species. There are two AtMTP8 orthologous genes in rice: the duplicated gene pair OsMTP8.1 and OsMTP8.2. Both proteins are localized at on the vacuole and are involved in Mn tolerance, similar to what is described for AtMTP8. However, decreased expression or loss-of-function of both transporters results in lower Mn concentrations in roots, but not in shoots, indicating that they may work differently than AtMTP8 regarding its role in controlling shoot translocation (Chen et al., 2013; Takemoto et al., 2017). Interestingly, OsMTP8.1 seems to be up-regulated by Fe deficiency, similar to AtMTP8 (Figure 1D). ### ROOT VACUOLAR COMPARTMENTALIZATION IMPACTS THE GRAIN IONOME Recent work has clearly shown that root vacuolar transporters can also affect mineral accumulation in grain. In rice, OsHMA4 is the causative gene of high grain Cu phenotype found in some accessions. Loss-of-function or natural variants with decreased OsHMA4 function result in increased Cu concentration in shoots and grains, as well as decreased concentration in roots and in root cell sap. Thus, OsHMA4 detoxifies Cu into root vacuoles, which decreases Cu translocation to shoots and grains (Huang et al., 2016). Similarly, the rice tonoplast-localized OsABCC1 transporter was shown to detoxify arsenic (As) by transporting As(III)-phytochelatin into vacuoles. The osabcc1 mutants have increased As sensitivity and As accumulation in grains (Song et al., 2014). In Arabidopsis, the duplicated pair AtABCC1/AtABCC2 also has a similar role (Song et al., 2010), again indicating that there is conservation of function in distantly related species. Moreover, OsHMA3 natural variation was clearly linked to high/low Cd in rice grains (Yan et al., 2016). The vacuolar iron transporter (VIT) family also deserves attention. In Arabidopsis, the AtVIT1 gene is key for correct distribution of Fe within seeds (Kim et al., 2006). Interestingly, rice has two genes, OsVIT1 and OsVIT2, which are involved in Fe storage in flag leaves. Intriguingly, high Fe (a common condition in lowland rice) up regulates OsVIT2 in roots, indicating that rice plants might have a mechanism to avoid Fe toxicity using root vacuolar compartmentalization. Moreover, OsVIT1/OsVIT2 also seem to regulate Fe distribution in seeds (Zhang et al., 2012), and endosperm-specific over-expression of the orthologous wheat gene TaVIT2 results in increased Fe content in wheat grains (Connorton et al., 2017). Based on that, we expect that Arabidopsis mutants and/or natural variants with weak alleles for AtMTP3, AtMTP8, AtFPN2, and AtHMA3 would have higher concentrations of their respective substrates in seeds. Indeed, AtMTP3-RNAi plants show increased Zn concentrations in whole inflorescences and siliques, and marginal increase in seeds (Arrivault et al., 2006). Under Mn sufficient conditions, loss-of-function mtp8 mutants and WT showed similar or slightly increased Mn concentration in seeds, whereas under Mn deficient conditions the mutants had decreased concentration compared to WT (Chu et al., 2017; Eroglu et al., 2017). These results suggest that in the presence of Mn, the lack of vacuolar root transporter allow more Mn to be translocated to seeds, compensating for decreased sink strength for Mn in the mtp8 mutant, which is apparent under Mn deficient conditions (Eroglu et al., 2017). It would be interesting to have data on Mn and Zn concentration in seeds of mtp8 mutants and AtMTP3-RNAi plants grown under high concentration of each element. Moreover, mutants or natural variants for AtHMA3 and AtFPN2 did not have their seed metal concentration evaluated (Morel et al., 2009; Morrissey et al., 2009; Chao et al., 2012), and no data for seeds of these plants is available in the Ionomics Hub database<sup>1</sup> . It is important to note that available evidence indicates that vacuolar compartmentalization is one of the checkpoints controlling the seed ionome, but by no means the only one. Depending on the element and its chemical speciation, other transporters expressed throughout the plant could contribute to the regulation of translocation to developing seeds (Sperotto et al., 2012; Punshon et al., 2018). An interesting example is highlighted by natural variation in AtHMA3, which is linked to species-wide Cd concentration variation in leaves but not to Zn and cobalt (Co) variation to the same extent, despite being able to transport both (Morel et al., 2009; Chao et al., 2012). One possibility is that elements such as Zn might be more tightly regulated, and thus variation in vacuolar transporter activity in roots might be compensated by other transporters (Chao et al., 2012). Thus, it remains to be tested the extent to which vacuolar compartmentalization is important to seed accumulation, which elements are most impacted, the transporters involved in this regulation, and how that varies in different species. ## CONCLUSION Biofortification of grains has been a long sought goal on the plant nutrition field, especially for Fe and Zn, the two most commonly lacking minerals in the human diet (Sperotto et al., 2012; Ricachenevsky et al., 2015). Arsenic is also a problem in rice, since it can accumulate in grains to harmful levels for human consumption (Punshon et al., 2017). Root vacuolar compartmentalization works in concert with other checkpoints to control elemental translocation to the shoots and, consequently, to the grains. Therefore, we should expect that mutants and/or accessions with loss-of-function alleles coding root vacuolar transporters have increased concentrations of the respective elements in the xylem sap, and potentially increased concentrations in seeds. We conclude that (1) tonoplast-localized root transporters can fine-tune symplastic concentrations of ions, together with apoplastic barriers and influx/efflux transporters; (2) plants are likely to tolerate changes in vacuolar storage capacity without strong changes in phenotype, since natural variation harbors loss-of-function alleles; and (3) orthologous genes in distantly related species might be hotspots of genetic variation (Morrissey et al., 2009; Chao et al., 2012; Huang et al., 2016; Yan et al., 2016). Thus, vacuolar transporters in roots are good candidates to search for interesting alleles and for engineering both shoot and seeds' ionome for biofortification and nutritional quality. <sup>1</sup>www.ionomicshub.org ### AUTHOR CONTRIBUTIONS fpls-09-00412 March 29, 2018 Time: 16:46 # 5 FR, JF, and RS wrote the manuscript. FR and AdAJ drew figures and presentation of previously published public data. All authors approved the final manuscript. ### REFERENCES ### FUNDING Authors were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Proc. Natl. Acad. Sci. U.S.A. 112, 10533–10538. doi: 10.1073/pnas.15076 91112 in iron-dependent nickel detoxification in Arabidopsis thaliana roots. J. Biol. Chem. 281, 25532–25540. doi: 10.1074/jbc.M601062200 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Ricachenevsky, de Araújo Junior, Fett and Sperotto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # The Nicotianamine Synthase Gene Is a Useful Candidate for Improving the Nutritional Qualities and Fe-Deficiency Tolerance of Various Crops #### Tomoko Nozoye1,2 \* <sup>1</sup> Center for Liberal Arts, Meiji Gakuin University, Kanagawa, Japan, <sup>2</sup> Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan With the global population predicted to grow by at least 25% by the year 2050, the sustainable production of nutritious foods will be necessary for human health and the environment. Iron (Fe) is an essential nutrient for both plants and humans. Fe is poorly soluble, especially at high pH levels, at which it is difficult for living organisms to accumulate sufficient Fe. In plants, Fe deficiency leads to low yield and poor nutritional quality, as it significantly affects chlorophyll synthesis. Fe deficiency is a worldwide agricultural problem that is especially serious in soils with a high pH, such as calcareous soils, which comprise approximately 30% of cultivated soils worldwide. Genetic improvements in crops that can tolerate Fe deficiency will be required to meet the demands for crop production and could ultimately contribute to the amelioration of global warming. Nicotianamine (NA) is an Fe chelator in plants that is involved in metal translocation in the plant body. In mammals, NA inhibits angiotensin I-converting enzyme, which plays a key role in blood pressure control. It was recently shown that the enhancement of NA production using nicotianamine synthase is useful for increasing not only NA but also Fe and Zn levels in crops such as rice, soybean, and sweet potato. Additionally, these plants showed Fe-deficiency tolerance in calcareous soil. These results suggested that NAS overexpression simultaneously improves food quality and increases plant production. This review summarizes progress in generating crops overexpressing NAS. Keywords: calcareous soil, iron (Fe), zinc (Zn), Fe deficiency, nicotianamine (NA) ### INCREASING Fe DEFICIENCY TOLERANCE COULD CONTRIBUTE TO FOOD SECURITY AND AMELIORATE GLOBAL WARMING Iron (Fe) is an essential nutrient for virtually all living organisms. Under aerobic conditions, Fe is oxidized to Fe(III) compounds, and their solubility in water is poor. Therefore, most Fe is not available to plants, although mineral soils contain 6% Fe by weight. Plants suffering Fe deficiency show leaf chlorosis, and their yield and nutritional quality are impaired dramatically ### Edited by: Alexander Arthur Theodore Johnson, University of Melbourne, Australia #### Reviewed by: Giacomo Cocetta, Università degli Studi di Milano, Italy Wricha Tyagi, Central Agricultural University, India \Correspondence: Tomoko Nozoye [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 03 December 2017 Accepted: 28 February 2018 Published: 27 March 2018 #### Citation: Nozoye T (2018) The Nicotianamine Synthase Gene Is a Useful Candidate for Improving the Nutritional Qualities and Fe-Deficiency Tolerance of Various Crops. Front. Plant Sci. 9:340. doi: 10.3389/fpls.2018.00340 235* (Marschner, 1995). This problem is exacerbated in soils with a high pH, such as calcareous soils, and is one of the major problems for crop production. Calcareous soils comprise approximately 30% of the cultivated soils worldwide (Chen and Barak, 1982). As the world population continues to increase, it is predicted that we will need 1.5 times more food in 2050 (High Level Expert Forum - How to Feed the World in 2050, 2009). It will be necessary to increase food production to meet this demand. However, the recent increase in atmospheric CO<sup>2</sup> levels is causing climate change, and it will be difficult to expand the area occupied by cultivated land by removing forests, which already contribute 2% of the CO<sup>2</sup> emissions (Intergovenmental Panel on Climate Change [IPCC], 2007). Problem soils, including calcareous soil, comprise 67% of the land globally (Food and Agriculture Organization of the United Nations). Improvements in plant growth in calcareous soils have great potential to increase the production of plant biomass and reduce atmospheric CO<sup>2</sup> levels, which will ultimately contribute to ameliorating global warming (Conway, 2012; Schroeder et al., 2013). In addition, Fe is necessary for human health, and its deficiency causes anemia, an easily identified disease that is a serious health problem, especially in developing countries (Welch and Graham, 2004). Ultimately, Fe in the human diet comes from plant uptake from the soil. Therefore, biofortification, i.e., increasing the Fe level in food plants, would improve human health. Appropriate target levels of Fe might differ according to the crop and target countries, as food cultures differ. For a rice-based diet, the target concentration of Fe was estimated to be 14.5 µg/g dry weight (DW) in polished rice grains, which is more than twice the present amount in rice (6 µg/g DW) (Hotz and McClafferty, 2007; Johnson et al., 2011). Therefore, genetically modified crops that can tolerate Fe deficiency while taking up sufficient Fe from calcareous soils would have a great impact on food security and contribute to ameliorating global warming. ### Fe ACQUISITION STRATEGIES IN PLANTS To acquire sparingly insoluble Fe from soil, plants have evolved two main strategies to acquire soil Fe (Marschner et al., 1986). Higher plants, not including graminaceous plants, which include soybean and sweet potato, are categorized as Strategy I plants, which reduce Fe(III) to Fe(II) by ferric-chelate reductases, and then take up Fe(II) via ferrous iron transporters IRT1 (Eide et al., 1996; Robinson et al., 1999; Vert et al., 2002). By contrast, graminaceous plants, including important staple crops such as rice, barley, and maize, are categorized as Strategy II plants, which produce and secrete Fe(III) chelators called mugineic acid family phytosiderophores (MAs) from their roots via the TOM1 transporter (Nozoye et al., 2011, 2013) and solubilize sparingly soluble Fe(III) in the rhizosphere (Takagi, 1976). Nicotianamine (NA) is a non-proteinogenic amino acid that was first found in tobacco (Noma et al., 1971). NA chelates many metal cations, including Fe, zinc (Zn), copper (Cu), and manganese (Mn) (Beneš et al., 1983; Murakami et al., 1989; von Wirén et al., 1999). NA exists in all plants examined so far, including Strategy I and II plants (Hell and Stephan, 2003; Takahashi et al., 2003; Schuler et al., 2012), and plays an important role in the internal transport of metal nutrients (Mori et al., 1991; Kawai et al., 2001; Hell and Stephan, 2003; Takahashi et al., 2003; Suzuki et al., 2006; Schuler et al., 2012). In graminaceous plants, NA also serves as an intermediate for the biosynthesis of MAs (Takagi, 1976; Mori and Nishizawa, 1987; Shojima et al., 1990). NA synthase (NAS) converts three molecules of S-adenosyl methionine into NA (Shojima et al., 1989, 1990; Higuchi et al., 1995). NAS genes were first isolated from barley and have subsequently been cloned from several plants species, including Arabidopsis, barley, rice, and maize (Herbik et al., 1999; Higuchi et al., 1999, 2001; Suzuki et al., 1999; Mizuno et al., 2003). Rice possesses three members: OsNAS1-3. OsNAS1, and OsNAS2 are mainly expressed in Fe-deficient roots and shoots, whereas OsNAS3 is also expressed in Fe-sufficient shoots (Inoue et al., 2003). It was suggested that all three have important roles in NA production under Fe-deficient conditions, although their roles might differ slightly. ### NICOTIANAMINE IS ALSO AN ATTRACTIVE FUNCTIONAL COMPONENT IN HUMAN HEALTH In mammals, NA inhibits angiotensin I-converting enzyme (ACE), which plays a key role in blood pressure control (Kinoshita et al., 1993). ACE plays a role in the renin–angiotensin system in the maintenance of blood pressure and fluids, as well as electrolyte homeostasis (Re, 2004). ACE inhibitors are widely used as antihypertensive agents (Chirumamilla et al., 2001; Re, 2004) The oral administration of NA causes ACE inhibitory activity in vitro and antihypertensive effects in spontaneously hypertensive rats; moreover, the strength of ACE inhibition is correlated with NA content (Izawa et al., 2008). The inhibitory activity of NA against ACE is very strong (Kinoshita et al., 1993; Kataoka, 2005). Almost all vegetables contain more than 44 µg/g DW NA, which has the ability to inhibit ACE activity by more than 60–70% (Izawa and Aoyagi, 2012). In addition, NA from pumpkin not only improves hypertension, but also long-term memory function (Takada, 2011). In fact, brain-penetrating ACE inhibitors such as captopril reduce the incidence of Alzheimer's disease in elderly hypertensive patients (Ohrui et al., 2004). Therefore, increased intake of NA through the diet could be effective for primary prophylaxis of hypertension and Alzheimer's disease. ### TRANSGENIC APPROACH TO INCREASING NA IN PLANTS Several reports have described transgenic plants generated by introducing the NAS gene (Table 1). The concentration of endogenous NA differs among crops (Izawa et al., 2008; Izawa and Aoyagi, 2012). The antihypertensive effect of NA was first identified in soybean (Kinoshita et al., 1993), which contains the highest amount of NA among the crops examined thus far (Izawa et al., 2008). In agreement with the endogenous NA level, the NA concentration in transgenic soybean was highest among the HvNAS1-overexpressing plants. The NA concentration in the transgenic soybean was increased to 768.1 µg/g DW in the seeds under the control of the cauliflower mosaic virus (CaMV) 35S promoter, which was four times higher than in non-transgenic (NT) seeds (Nozoye et al., 2014a). In sweet potato, overexpression of HvNAS1 by the CaMV 35S promoter increased the NA concentration to 339.5 µg/g fresh weight (FW) in the leaves and 225.9 µg/g FW in the storage roots, which were 9.1 and 4.6 times higher, respectively, than in NT plants (Nozoye et al., 2017). In comparison, the NA concentration in HvNAS1-overexpressing tobacco (a dicot) by the CaMV 35S promoter was 78.9 µg/g FW in the leaves, which was 8.7 times higher than in NT plants (Kim et al., 2005). It was suggested that NAS genes are separated into two clusters between Gramineae and dicots and that the NASs in soybean and sweet potato were most similar in the dicot cluster (Nozoye et al., 2017). The NASs in soybean and sweet potato might have high enzymatic activity and produce more NA. The endogenous NA concentrations also differed among the tissues in rice (Table 1). The NA concentrations in leaves tended to be higher than that in the seeds. Consistent with the endogenous NA levels, the NA concentrations in leaves were also higher than those in seeds in NAS-overexpressing rice plants. Overexpression of HvNAS1 by the CaMV 35S promoter increased the NA concentration to 75.8 µg/g DW in the polished seeds, which was 10.6 times higher than in NT seeds (Masuda et al., 2009). Overexpression of HvNAS1 by the OsActin1 promoter increased the NA concentration to 30.3 µg/g DW in the polished seeds, which were 16 and 5.1 times higher, respectively, than in NT plants (Masuda et al., 2009). By overexpressing rice NAS genes (OsNAS1–3) in rice under the control of an enhanced CaMV 35S promoter, the NA concentration in rice seeds increased to 210 µg/g DW, which was 9.3 times higher than in NT seeds (Johnson et al., 2011). In comparison, by overexpressing OsNAS1 in rice under the control of the maize ubiquitin promoter, the NA concentration in rice leaves increased to 400 µg/g DW, which was 6.7 times higher than in NT leaves (Zheng et al., 2010). Using seed-specific expression of OsNAS1 under the control of the rice glutelin promoter, the NA concentration in rice seeds increased to 65 µg/g DW, which was 5.2 times higher than in NT seeds (Zheng et al., 2010). This concentration was slightly lower than that in NAS-overexpressing rice seeds under the control of ubiquitous promoters. Additionally, in these plants, the NA concentration in shoots was not different from that in NT plants. These results suggest that it is possible to achieve a greater increase in NA in seeds by enhancing NA mobilization and translocation from leaves (and roots) to seeds. The average increase (fold change) in NA concentration was 7.6 and did not differ significantly among the crops, suggesting that the amount of endogenous NA is not a factor that limits the NA concentration. The combined enhancement of NAS and NA transporters could further elevate the NA level in the edible parts of the plant. fpls-09-00340 March 24, 2018 Time: 13:56 # 3 TABLE 1 concentrations in NAS-overexpressing plants. ### ENHANCEMENT OF NA INCREASED THE Fe AND Zn CONCENTRATIONS IN PLANTS Nicotianamine plays an important role in metal transport in the plant body. It was suggested that NA is involved in the translocation of Fe and Zn into seeds in rice, Arabidopsis, tomato, and tobacco (Higuchi et al., 1996; Takahashi et al., 2003; Kim et al., 2005; Masuda et al., 2008, 2009; Schuler et al., 2012). Fe is readily oxidized and precipitated in the apoplasm of both roots and shoots. Therefore, Fe uptake from the apoplasm is important for plant growth. In the Arabidopsis double IRT1 and Nramp1 mutant, Fe was precipitated and accumulated in the apoplast of the roots, while the Fe concentration in shoots was dramatically reduced compared with NT (Castaings et al., 2016). In NASoverexpressing plants, the Fe and Zn concentrations were also increased (Table 1). NA might be involved in the mobilization of Fe and Zn in the apoplasm. In the seeds of HvNAS1 overexpressing soybean plants, the Fe and Zn concentrations increased to 110 and 65 µg/g DW, which were 2 and 1.45 times higher, respectively, than in NT plants (Nozoye et al., 2014a). In HvNAS1-overexpressing sweet potato, the Fe and Zn concentrations increased to 52.9 and 17 µg/g FW in the leaves and 15.1 and 3.5 µg/g DW in the storage roots, which were 3 and 3, and 2.1 and 3.5 times higher, respectively, than in NT plants (Nozoye et al., 2017). In HvNAS1-overexpressing tobacco, the Fe and Zn concentrations increased to 5.3 and 9.6 µg/g FW in the leaves, which were 5 and 2.3 times higher, respectively, than in NT plants (Kim et al., 2005). In HvNAS1-overexpressing rice via the CaMV 35S promoter, the Fe and Zn concentrations increased to 9 and 45 µg/g DW, respectively, in the polished seeds, which were 2 and 1.5 times higher than in NT seeds (Masuda et al., 2009). In contrast, in HvNAS1-overexpressing rice via the OsActin1 promoter, the Fe and Zn concentrations were 170 and 25 µg/g DW in the leaves and 5 and 40 µg/g DW in the polished seeds, respectively, which did not differ significantly from those in NT plants (Masuda et al., 2009). By overexpressing rice NAS genes (OsNAS1–3), the Fe and Zn concentrations in rice seeds increased to 81 and 91 µg/g DW, respectively, which were 3.5 and 2.2 times higher than in NT seeds (Johnson et al., 2011). In OsNAS1-overexpressing rice via the maize ubiquitin promoter, the Fe and Zn concentrations in rice seeds were increased to 28 and 120 µg/g DW, respectively, which were 2.3 and 5.5 times higher than in NT seeds (Zheng et al., 2010). Seed-specific expression of OsNAS1 under control of the rice glutelin promoter, increased the Zn concentration in rice polished seeds to 29.07 µg/g DW, which was 2.3 times higher than in NT seeds (Zheng et al., 2010); however, the Fe concentration was not altered in the polished seeds. In these plants, the Fe and Zn concentrations in leaves were increased to 18 and 30 µg/g DW, which were 1.8 and 1.9 times higher, respectively, than in NT leaves. As with the NA concentration, the Fe and Zn concentrations tended to be higher in soybean seeds; however, this difference was not significant compared to the NA concentration. In agreement, the increases in Fe and Zn concentrations were lower than those of the NA concentration (Table 1). The average increases in Fe and Zn were 2.2 and 2.3, respectively, whereas that of NA was 7.6. There might be a factor limiting the increases in the metals compared with NA. Because Fe and Zn are taken up from the soil via roots, modification of the uptake system might further increase Fe and Zn. It is also possible that the increased NA in NAS-overexpressing plants was not translocated in the plant body efficiently. There might be potential to increase Fe and Zn by changing the flow of NA in the plant body. ### NAS-OVEREXPRESSING PLANTS SHOWED TOLERANCE TO Fe DEFICIENCY Several NAS-overexpressing plants have been confirmed to tolerate Fe deficiency compared to NT plants (Lee et al., 2009; Nozoye et al., 2014a, 2017). The plant growth is dramatically reduced under Fe-deficient conditions. The plant heights and soil and plant analyzer development (SPAD) values (which represent the chlorophyll content) of NAS-overexpressing soybean and sweet potato plants were higher than those of NT plants when grown in calcareous soil with low Fe availability, suggesting that these transgenic plants were conferred tolerance to Fe deficiency. Under normal soil conditions, their growth did not differ. In rice plants, the Fe-deficiency tolerance of NAS-overexpressing rice plants in calcareous soil was not determined. HvNAS1-overexpressing rice exhibits enhanced NAS activity in Fe-deficient roots (Higuchi et al., 2001) and contains a higher amount of NA and deoxymugineic acid than NT plants in both roots and shoots (Masuda et al., 2009). Transgenic rice lines expressing barley NAS genes exhibit increased tolerance to low Fe availability in calcareous soil (Suzuki et al., 2008). Rice plants overexpressing OsIRO2, a transcription factor that enhances expression of Fe deficiency-inducible genes including OsNAS1 and OsNAS2, showed improved tolerance to low Fe availability in calcareous soil (Ogo et al., 2011). These results suggest that overexpression of the NAS gene in rice also enhances tolerance to Fe deficiency. It was recently suggested that NA may be involved in Fe homeostasis; enhanced NA production induced Fe deficiency signaling and mobilization of Fe in the plant body (Nozoye et al., 2014b,c). Since NA has the ability to chelate Fe, NA may enable the de-repression of Fe deficiency-inducible genes by drawing Fe from an unknown Fe-sensing mechanism, and further increase the NA and deoxymugineic acid (a primary MAs) levels. NA has long been considered a candidate long-distance Fe signaling molecule in both gramineous and dicot plants (Curie and Briat, 2003); however, this has not yet been proven. In rice, NAS overexpression positively modulates Fe homeostasisrelated genes (Wang et al., 2013). NA accumulation in Osnaat1 mutants triggers a constitutive Fe deficiency response (Cheng et al., 2007). In Arabidopsis, NA-over-accumulating plants showed an Fe-deficient phenotype and expressed Fe-inducible genes at higher levels than did NT plants; however, they also contained more Fe than did NT plants, suggesting that an increase in the NA apoplastic pool sequestered Fe, which controls plant Fe homeostasis (Cassin et al., 2009). The overexpression of ZINC-INDUCED FACILITATOR 1 (ZIF1) in Arabidopsis increased the amount of NA in the roots and shoots and led to Fe deficiency (Haydon et al., 2012). ZIF1 is a vacuolar membrane-localized putative transporter required for Zn tolerance that is hypothesized to transport NA from the cytoplasm into the vacuoles. Perturbing the subcellular distribution of NA may have profound effects on Fe with respect to subcellular distribution and inter-organ partitioning. In agreement with this phenomenon, it was revealed that AtYSL1 and AtYSL3, Fe-NA transporters, are required for proper longdistance Fe signaling (Kumar et al., 2017). A ysl1ysl3 doublemutant did not up- or down-regulate Fe deficiency-induced or -repressed genes, while it contained markedly low tissue levels of Fe compared to NT plants. These results suggest that NA may be involved in long-distance signaling to maintain Fe homeostasis. In NAS-overexpressing plants, the increased NA might induce Fedeficiency-inducible genes that contribute to conferring tolerance to Fe deficiency. ### CONCLUSION Overexpression of the NAS gene enhances NA levels in several crops, including crops in which endogenous NA is already high, such as soybean and sweet potato. Additionally, NAS overexpression enhances the Fe and Zn concentrations and confers tolerance to Fe deficiency in calcareous soil. The increase ### REFERENCES in NA tended to be higher in leaves than in seeds, while the increases in Fe and Zn were lower than those of NA. These results suggest the potential to increase NA, Fe, and Zn concentrations further in the edible parts of crops. Further analysis of NA translocation in the plant body will allow for improved engineering strategies not only to accumulate bioavailable Fe in edible parts, but also to increase the tolerance of plants to low Fe availability to meet the demands of plant production and to solve problems such as inadequate diet, food shortages, and global warming in the near future. ### AUTHOR CONTRIBUTIONS TN designed and wrote the manuscript. ## FUNDING This publication was supported by a grant-in-aid for Young Scientists (B) (Grant No. 15K18658) from JSPS KAKENHI (to TN) and by a grant from Uragami-zaidan (to TN). ### ACKNOWLEDGMENTS I thank Prof. Naoko K. Nishizawa for reading and commenting on the manuscript. Soil Sci. Plant Nutr. 22, 423–433. doi: 10.1080/00380768.1976.1043 3004 Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Nozoye. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Zinc and Iron Concentration as Affected by Nitrogen Fertilization and Their Localization in Wheat Grain Bal R. Singh<sup>1</sup> \, Yadu N. Timsina<sup>1</sup> , Ole C. Lind<sup>2</sup> , Simone Cagno2,3 and Koen Janssens<sup>3</sup> <sup>1</sup> Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Ås, Norway, <sup>2</sup> Centre of Environmental Radioactivity, Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Ås, Norway, <sup>3</sup> Department of Chemistry, University of Antwerp, Antwerp, Belgium Nearly half of the world cereal production comes from soils low or marginal in plant available zinc, leading to unsustainable and poor quality grain production. Therefore, the effects of nitrogen (N) rate and application time on zinc (Zn) and iron (Fe) concentration in wheat grain were investigated. Wheat (Triticum aestivum var. Krabat) was grown in a growth chamber with 8 and 16 h of day and night periods, respectively. The N rates were 29, 43, and 57 mg N kg−<sup>1</sup> soil, equivalent to 80, 120, and 160 kg N ha−<sup>1</sup> . Zinc and Fe were applied at 10 mg kg−<sup>1</sup> growth media. In one of the N treatments, additional Zn and Fe through foliar spray (6 mg of Zn or Fe in 10 ml water /pot) was applied. Micro-analytical localization of Zn and Fe within grain was performed using scanning macro-X-ray fluorescence (MA-XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The following data were obtained: grain and straw yield pot−<sup>1</sup> , 1000 grains weight, number of grains pot−<sup>1</sup> , whole grain protein content, concentration of Zn and Fe in the grains. Grain yield increased from 80 to 120 kg N ha−<sup>1</sup> rates only and decreased at 160 kg N ha−<sup>1</sup> g. Relatively higher protein content and Zn and Fe concentration in the grain were recorded with the split N application of 160 kg N ha−<sup>1</sup> . Soil and foliar supply of Zn and Fe (Zn + Fes+<sup>f</sup> ), with a single application of 120 kg N ha−<sup>1</sup> N at sowing, increased the concentration of Zn by 46% and of Fe by 35%, as compared to their growth media application only. Line scans of freshly cut areas of sliced grains showed co-localization of Zn and Fe within germ, crease and aleurone. We thus conclude that split application of N at 160 kg ha−<sup>1</sup> at sowing and stem elongation, in combination with soil and foliar application of Zn and Fe, can be a good agricultural practice to enhance protein content and the Zn and Fe concentration in grain. Keywords: nitrogen application, zinc and iron uptake, zinc and iron distribution in grain, wheat, LA-ICP-MS, MA-XRF ### INTRODUCTION Cereals are genetically low in Zn and Fe concentration, with reduced bioavailability (Graham et al., 2001; Cakmak, 2002). About half of the world cereal production come from soils low in plant available Zn (Cakmak, 2002), leading to poor quality of cereal grain with respect to Zn content. The situation is similar, concerning Fe deficiency in cereals. About one third of the developing countries' population and 10% of Americans and Canadians experience Zn deficiency or are at risk ### Edited by: Raul Antonio Sperotto, University of Taquari Valley, Brazil ### Reviewed by: Ümit Barı ¸s Kutman, Gebze Technical University, Turkey Lourdes Hernandez-Apaolaza, Universidad Autonoma de Madrid, Spain > \Correspondence: Bal R. Singh [email protected] ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 01 November 2017 Accepted: 22 February 2018 Published: 09 March 2018 #### Citation: Singh BR, Timsina YN, Lind OC, Cagno S and Janssens K (2018) Zinc and Iron Concentration as Affected by Nitrogen Fertilization and Their Localization in Wheat Grain. Front. Plant Sci. 9:307. doi: 10.3389/fpls.2018.00307 242 of it (Hotz and Brown, 2004), erasing the geographical and political boundaries. Every year Fe and Zn deficiency causes deaths of about 800000 children and 2.4% of global disabilityadjusted life years worldwide (DALYs), while the corresponding value of DALYs for Zn is 1.9% (Mutangadura, 2004). DALYs are calculated as the sum of years of life lost (YLLs) and the years lived with disability (YLDs) based on 291 causes and 20 age groups of both sexes. The consumption of white flour made predominantly from endosperm of wheat grain discarding bran in the milling process has even worsened the degree of Fe and Zn malnutrition. This is because Fe and Zn accumulate in higher concentrations in the embryo and aleurone layer than in endosperm of a wheat grain (Šramková et al., 2009; Cakmak et al., 2010). Hence, the consumption of whole grain wheat rather than white wheat flour has been advocated to increase the daily Fe and Zn intake. Nitrogen fertilization is known not only to increase wheat grain yield but also to facilitate the uptake of Fe and Zn in wheat grain (Cakmak et al., 2010; Shi et al., 2010). The uptake and transport of Fe and Zn to grain is probably facilitated by metal chelating compounds (Kutman et al., 2010), such as 2-deoxymugineic acid (DMA) mainly for the translocation of Fe and Zn from flag leaves to grain in wheat (Barunawati et al., 2013). Kutman et al. (2011) reported that N nutrition is critical in both the uptake and translocation of Zn and Fe to wheat grain and they showed that at high N rate, nearly 80% and 60% of total shoot Zn and Fe, respectively, were harvested with grain. Improving N status of plants from low to sufficient resulted in threefold increase in shoot Fe content of wheat plants (Aciksoz et al., 2011) Similarly, Erenoglu et al. (2011) demonstrated that N is a critical player in the uptake and accumulation of Zn in plants and thus deserves special attention in biofortification of food crops with Zn. Depending on N supply, Zn remobilization from pre-anthesis sources provided almost all grain Zn, when the Zn supply was withheld at anthesis (Kutman et al., 2012). Cakmak et al. (2010) found co-localization of protein, Fe and Zn in embryo and aleurone layer of wheat grain, indicating that the protein rich grains accumulate higher amount of Zn and Fe in wheat grain. Increasing Zn and N supply had a major impact on Zn accumulation in the endosperm, reaching concentrations higher than the current breeding targets (Persson et al., 2016). Cakmak et al. (2010) suggested the positive role of soil and foliar applied Zn and Fe in increasing respective metal concentrations in durum wheat grain and also claimed that increased activity of Zn and Fe in the source (flag leaf and stem) during grain filling could be increased by additional Zn and Fe application through soil or by foliar application. Habib (2012) showed that joint Zn-Fe application could increase in grain concentration more than with their separate application. However, the concentrations of Zn and Fe depend on the size of wheat grains (Velu et al., 2011) and number of grains per spike (Nowack et al., 2008). Haslett et al. (2001) and Timsina (2014) demonstrated the role of phloem transport of Zn in wheat plants by performing stem girdling, and they showed that <sup>65</sup>Zn supplied on upper leaf was transported to lower leaves and root tip. The concentration of minerals vary within a grain, depending on its portions. For example, wheat endosperm contains about 15 mg kg−<sup>1</sup> Zn, while germ and aleurone holds about 150 mg kg−<sup>1</sup> Zn (Šramková et al., 2009). By using laser ablationinductively coupled plasma-mass spectrometry (LA-ICP-MS), Wang et al. (2011) depicted higher concentration of Zn in the aleurone layer and crease vascular tissue with decreasing gradient of Zn from crease vascular tissue to endosperm, suggesting that translocation of Zn toward the endosperm occurred through the crease vascular tissue. Moreover, protein rich grain accumulated higher amount of Zn and Fe in wheat than low protein grain (i.e., Fe = 71 mg kg−<sup>1</sup> and Zn = 57 mg kg−<sup>1</sup> vs. Fe = 36 mg kg−<sup>1</sup> and Zn = 30 mg kg−<sup>1</sup> ) (Ozturk et al., 2009). This showed that higher protein or nitrogen content favors the accumulation of Zn and Fe in wheat grain (Peleg et al., 2008; Ozturk et al., 2009; Kutman et al., 2010). In spite of the available literature on the role of N on Zn and Fe uptake by plants, information on the optimum rate and time of N application, and its effect on Zn and Fe uptake under varying levels of micronutrients in the soil are scanty. Similarly, the localization of these micronutrients in grains is not fully understood. We hypothesized that (i) N fertilization increases protein yield components of wheat and the concentration of Fe and Zn in grain, (ii) foliar Zn and Fe spraying increases their concentration in wheat grain, and (iii) micro-analytical techniques can provide information on the location of Zn and Fe in wheat grain. To test these hypotheses, we investigated the interactive effect of N, Zn, and Fe on grain yield, protein content and nutrient concentration in a pot experiment conducted in an environmentally controlled growth chamber. In addition, we investigated the distribution of Fe and Zn in selected wheat grains by using scanning macro-X-ray fluorescence (MA-XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ### MATERIALS AND METHODS ### Growth Media Artificially prepared growth media, according to the OECD 207 guideline (OECD, 1984), were used for growing plants. They consisted of 80% sand (<2 mm), 10% peat (<4 mm), and 10% kaolin on a dry weight basis. In the absence of sphagnum peat, unfertilized natural peat, produced by Econova Garden AB, Sweden, was used. Air dried peat was sieved through 4 mm wire mesh and average moisture content was determined by drying nine representative samples in an oven at 105◦C for 24 h for the correction of moisture content in peat. Average moisture content varied from 41 to 48% depending on peat delivery bags. To maintain the pH of growth media at 6.5 ± 0.2, CaCO<sup>3</sup> was mixed at rates from 0.5 to 5 g per 100 g growth media and the amount of lime required was calculated from the liming curve obtained. Hand mixed growth media and lime were prepared. The homogeneous growth media mixture was filled in 3-l plastic pots containing 2015 g mixture (dry weight) in each pot. ### Experimental Design and Fertilizer Rates The experiment was set up as a complete randomized factorial design (Table 1). and It consisted of two major treatment factors: N treatments and Zn-Fe treatments. The experiment was further Where, N-treatments: Single soil application of N at sowing: N1N80, single application of N at sowing equivalent to 80 kg N ha−<sup>1</sup> mixed wit growth media. N1N120, single application of N at sowing equivalent to 120 kg N ha−<sup>1</sup> mixed with growth media. N1N160, single application of N equivalent to 160 kg N ha−<sup>1</sup> mixed with growth media. Split application of N: N2N120, Split application of N equivalent to 120 kg N ha−<sup>1</sup> . 70% (equivalent to 84 kg N ha−<sup>1</sup> ) of allocated N was applied at sowing time and 30% (equivalent to 36 kg N ha−<sup>1</sup> ) at the beginning of stem elongation. N2N160, Split application of N equivalent to 160 kg N ha−<sup>1</sup> . 70% (equivalent to 112 kg N ha−<sup>1</sup> ) of allocated N was applied at sowing time and 30% (equivalent to 48 kg N ha−<sup>1</sup> ) at the beginning of stem elongation. Zn–Fe treatments: Soil application of Fe and Zn at sowing: Zn, Zn mixed with soil at sowing. Zn + Fe, Zn and Fe mixed with growth media at sowing. Fe, Fe mixed with soil at sowing. Soil plus foliar application of Fe and Zn at booting stage: Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> , application of Zn at sowing plus 30% of growth media applied Zn as foliar spray. (Zn + Fe)<sup>s</sup> <sup>+</sup> <sup>f</sup> , application of Zn and Fe at sowing plus 30% of growth media applied zinc and iron as foliar spray. Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> , application of Fe at sowing plus 30% of growth media applied iron as foliar spray. divided into two groups: (i) experiments with growth media application of all treatment factors (5 N treatments × 3 Zn–Fe treatments = 15 growth media treatments) and (ii) experiments with growth media l plus foliar spray of Zn and Fe (2 N treatments × 3 Zn–Fe treatments = 6 growth media plus foliar treatments) (Table 1). Both treatment factors were incorporated into the same experiment to see the combined effect of them. Among the five N-treatments, three were single N application to growth media before sowing at the rates equivalent to 80, 120, and 160 kg N ha−<sup>1</sup> , and two were split N applications at the rates equivalent to 120 kg N ha−<sup>1</sup> and 160 kg N ha−<sup>1</sup> . In split application treatments (N120 and N160 kg N ha−<sup>1</sup> ), 70% N was applied at sowing and 30% at the stem elongation phase. Similarly, the three Zn–Fe treatments included Zn, Fe, and Zn + Fe. The rate of Zn and Fe application at sowing was 10 mg kg−<sup>1</sup> of growth media. Foliar spray of Zn and Fe (equal to 6 mg pot−<sup>1</sup> ) was made at booting stage in the two N treatments (single application and split application of 120 kg N ha−<sup>1</sup> . This was done to assess the effect of foliar spray of Zn and Fe on wheat grain yield and Zn and Fe concentration. Since the artificially made growth media used supplied all nutrients required for plant growth, the need of having a control pot was not felt. All basic nutrients and the treatment factors (N, Zn, and Fe) were applied in deionized water solution. Powdered calcium carbonate was mixed with growth media to maintain soil pH at 6.5 ± 0.2. The treatment combinations and rates and sources of N fertilizers and micronutrients are presented in Tables 1, 2, respectively. The solution volume of all nutrients was fixed to 25 ml, which was later taken into account while watering the growth media after sowing. All added nutrients and lime were mixed manually to the growth media to get a homogeneous distribution. The second dose of nitrogen in split nitrogen treatments (N2N120 and N2N160), amounting to 30% of the total N, was added at the beginning of stem elongation and watered immediately, so that N could spread properly. A handheld sprayer was used. The sprayed solution of 10 ml water per pot contained 6.0 mg of Zn as zinc sulfate and 6.0 mg of Fe Fe-EDTA mixed with DP-Klebemiddel surfactant with a concentration of 0.5 ml per one liter solution. Spraying was done after complete emergence of flag leaf at booting stage and 10 mL solution was sprayed several times to ensure that the whole solution was effectively sprayed on plant leaves. ### Plant Growth and Harvesting Wheat plants were grown in a control growth chamber at about 21◦C. The duration of day and night length was 8 and 16 h, respectively. The source of light was halogen metal halide lamps by POWERSTAR HQI-BT 400W/D. The test plant was a hard red winter wheat variety "Krabat" used by farmers since 2011 which is claimed to be medium early in growth period, high yielding with good agronomical characteristics, medium protein, relatively good disease resistance and baking quality. Twenty seeds were sowed in each pot, which after 1 week were thinned to eight plants. While watering for the first time, the amounts of water contributed by peat and other liquid nutrients were taken into consideration to maintain moisture at 60% of field capacity throughout the growth period. It was achieved by weighing the pots with soil mixture and plants regularly and adding water to compensate the weight loss. Plant were harvested at maturity by cutting each spike separately, and these were kept in bags for each pot. After removal of spikes, straw was cut just at the base of first node. Grain and straw yields were recorded after oven drying at 75◦C for 48 h. Wheat grain and straw were ground in a ball mill (Retsch MM301), with ball and container walls of zirconium to avoid sample contamination. However, for the sake of brevity, only grain yield, protein, Zn, and Fe concentration in grain are reported in this paper. ### Chemical Analysis Total N in grain nitrogen was analyzed by dry combustion as described by Bremner and Mulvaney (1982). The whole grain protein (WGP) was obtained by multiplying the total N by a factor of 5.70 (ISO, 2009). All solutions were added to growth media before sowing at volume 25 ml. Rows with bold letters represent for treatment factors. About 0.2 g of ground wheat flour was digested in 5 ml conc. HNO<sup>3</sup> for about 2 h in ultra clave microwave reactor (MLS-MILESTONE, ultra-CLAVE III) at 250◦C and at 160 bar pressure. The digested samples were diluted to 50 ml by adding double de-ionized water (B-pure, Barnstead). Three Standard Reference Materials (SRM) (SRM1567a wheat flour) and 5 method blanks (5 ml HNO<sup>3</sup> solution) were also digested along with grain samples. Concentrations of Fe and Zn were analyzed by an inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin-Elmer Optima 5300 DV) in wheat samples, SRM, and method blanks. The lower detection limits (LOD's) and lower quantification limits (LOQ's) were determined for the concentration of Fe and Zn in the method. Measured concentrations of Fe and Zn in all samples were higher than LOD's (Average of blanks plus 3 times the SD) and LOQs (Average of blanks plus 10 times the SD). The accuracy of analytical method was determined by the analysis of three replicates of standard reference materials (SRMs 1567a wheat flour). The measured concentrations of Fe and Zn in SRMs were in accordance with the certified concentration limits and the RSD was <5%. ### Localization of Fe and Zn in Wheat Grain Six selected wheat grains from different treatments containing relatively high Zn (42–99 mg/kg) and Fe (44–115 mg/kg) concentration were used. These grain samples were collected from different pots showing higher Zn and Fe concentration and mostly from foliar experiment and thus here the effect of N on Zn and Fe location was not clearly investigated. At first, the Environmental scanning electron microscope with dispersive X-ray spectrometry (ESEM-EDS) technique, available at the Norwegian University of Life Sciences (NMBU), Norway, was used for observation of grain morphology and element distribution. However, the limit of detection of ESEM-EDS (0.1% w/w) was not sufficiently low to detect and quantify Zn and Fe in the expected concentrations. For trace element 2D distribution analysis, Scanning macro- X-ray fluorescence (MA-XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) were performed at the University of Antwerp, Belgium. Each seed was sliced in two parts. Slicing of grains was done with a razor blade that was cleaned with ethanol prior to use. The half grains were measured with no additional sample preparation. In the case of MA-XRF, they were mounted by means of adhesive tape on a diapositive frame positioned vertically on sample holder. The measurement, after verifying the sample-sourcedetector distance, was performed in an entirely non-invasive way. In the case of LA-ICP-MS, they were introduced mounted horizontally on plasticine on the floor of the sample chamber, which was closed and purged. Detailed photographs of the surface were acquired by means of the instrument software, and the line profiles were drawn and ablated on the basis of those. MA-XRF was performed using a non-commercial selfassembled Scanning macro- X-ray fluorescence (XRF), with the setup named instrument C (Alfeld et al., 2011). The elemental maps were recorded with a step size of 25-µm and dwell time of 400 ms per point with tube settings of 50 kV and 1.0 mA (35 W). The beam size at the focal point was approximately 50 µm. XRF maps were obtained for each element detectable in the grains. The most relevant elements were Fe, Zn, and K, whose distribution is shown in Figure 6. For a more detailed analysis of these elements and their co-occurrence in the grain, the following technique was used. LA-ICP-MS was performed with a New Wave NWR193 ArF excimer laser and a Varian 7700 quadrupole ICP-MS. The ablation of sample was performed in helium gas (He) and transported to the plasma in argon gas (Ar). The flow rate was set to 0.4 l/min for carrier gas and 0.7 l/min for makeup gas. The forward power was set to 1350 watt. The line scan was executed at the speed of 10 µm/s with dimension more than 3 mm length and 100 µm beam diameter. The repetition rate of scan was 20 Hz at 90% energy capacity. The fluence was maintained at approximately 8 J/cm<sup>2</sup> . The laser warm up at the beginning of scan lasted until 20 s and washout begin after about 290 s and lasting until 350 s. This generated gross element counts, which were further refined in the following way. Background counts were collected along the scan line before and after the wheat grain (which was located in the middle of a scan line). Net counts were determined by subtracting from the gross counts the average background counts (after removal of outliers) for each element (K, Fe, Zn). Finally, the normalized counts of Zn and Fe were determined by dividing their respective net counts with net counts for K. Potassium, (39K) was used as the normalizing element since it occurs in a more evenly distributed way throughout the grain, and particularly in the crease, as made visible by MA-XRF in Figure 6D. The use of K-normalized counts for Fe and Zn, helps evidencing any real increase of either element in spots/areas of the grain, independently from total ion count and surface/positioning effects. ### Statistical Analysis The analysis of variance (ANOVA) was performed by twoway ANOVA and the relations between variables were analyzed by regression model using Minitab 16. During the regression analysis, data for independent variables were centered on their average value when necessary. For centering of data, each observed value was subtracted from the average of the respective variable. The comparison between all treatments, considering the interaction of treatment factors and main effects of N- and Zn– Fe- treatments, was carried out by Tukey comparison. In all cases, data were analyzed considering 5% level of significance (p = 0.05). ### RESULTS ### Grain Yield The grain yield pot−<sup>1</sup> increased while increasing N rate from 80 to 120 kg N ha−<sup>1</sup> , but decreased when N was increased to 160 kg N ha−<sup>1</sup> . A similar trend was also observed for split application of N from 120 to 160 kg N ha−<sup>1</sup> . The single application of 120 kg N ha−<sup>1</sup> at sowing (N1N120) and split application of 160 kg N ha−<sup>1</sup> at sowing and stem elongation (N2N160) resulted in the highest yield (Figure 1). Likewise, the growth media application of Zn produced higher yield than growth media applied Fe and Zn + Fe, particularly at the application rate of 160 kg N ha−<sup>1</sup> at sowing (Figure 1). The growth media plus foliar application (Zns+<sup>f</sup> or Fes+<sup>f</sup> ) for Zn and Fe, single or together, increased the grain yield in comparison to their growth media application at sowing. The single application (N1N120) of 120 kg N ha−<sup>1</sup> resulted in significantly higher yield (3.02 ± 0.04 g pot−<sup>1</sup> ) than the split application (N2N120) of 120 kg N ha−<sup>1</sup> (2.87 ± 0.04 g pot−<sup>1</sup> ) (Table 3) in all combinations of Zn and Fe, except for growth meida plus foliar spray of Zn. The Zn-Fe treatment (Zn + Fe) did not show a significant difference in mean grain yield for experiments with growth media l plus foliar application (Zn + Fe<sup>s</sup> + <sup>f</sup> ) of Zn and Fe (p = 0.107). ### Whole Grain Protein (WGP) The growth media and foliar application (Zns+<sup>f</sup> or Fes+<sup>f</sup> ) resulted in significantly higher wheat grain protein (WGP) as compared to growth media application (Zn or Fe) (p = 0.021) at single as well as split N equivalent to 120 kg N ha−<sup>1</sup> (Table 4). Irrespective of N treatments, average increase of WGP for growth media l plus foliar application of Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> , Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> and Fs + fe was 8.4, 6.5, and 7% as compared to their growth media applied rate (Zn + Fe, Zn, Fe). Among all treatments, the growth media plus foliar application of Zn + Fe at split application of 120 kg N ha−<sup>1</sup> (Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> at N2N120) produced the highest protein rich grains (8.96 ± 0.133%), suggesting that split N application is a better method to achieve higher protein in wheat grain. Zinc in combination with single growth media application of 120 kg N ha−<sup>1</sup> (Zn at N1N120) gave the lowest protein content (7.95 ± 0.06%) (Table 4). ### Iron Concentration in Wheat Grain Iron concentration in grain was significantly affected by growth media supplied N (p < 0.001). The increasing rate of N from N1N80 to N1N160) resulted in significantly higher Fe concentration in grain when Fe was applied alone (Figure 2). The growth media and foliar application (Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> and Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ) of Fe increased the Fe− concentration in wheat grain significantly (p < 0.001) as compared to their growth media applied rate (Fe and Zn + Fe). Both growth media (Zn + Fe) or growth media plus foliar application (Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ) of Zn + Fe showed higher Fe- concentration in wheat grain, but a significantly higher Fe-concentration was achieved only with Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> at N1N120 (Figure 3). ### Zinc Concentration in Wheat Grain Grain Zn-concentration responded significantly to the main effect of N-treatment (p < 0.001). When N rate at sowing increases from 80 to 120 kg N ha−<sup>1</sup> (N1N80 to N1N120), the concentration of Zn in grain tends to decrease (Figure 4). For instance, single application of 120 kg N ha−<sup>1</sup> (N1N120) and split application of 160 kg N ha−<sup>1</sup> (N2N120) showed about 10% less Zn as compared to single application of 80 and 160 kg N ha−<sup>1</sup> (N1N80 or N1N160). All other combinations of treatments showed generally the same Zn concentration. In the experiments with growth media plus foliar spray of Zn and Fe (Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> and Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ), Zn- concentration in wheat grain increased significantly (p < 0.01), but was not affected by N- treatments and the interaction between N- and Zn–Fetreatments. The increase in the grain Zn-concentration in foliar sprayed treatments (Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> and Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ) was higher than with Zn or Zn + Fe treatments at N1N120 or N2N120, but a significantly higher Zn- concentration was achieved only with Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> at N1N120 (Figure 5). ### Relationship Between Fe- and Zn-Concentration and Grain Yield Parameters A multiple linear regression including all measurements for 84 growth pots provided a valid relationship (p < 0.001) between Feconcentration in grain and the total grain weight (TGW), number FIGURE 1 \| Grain yield at N when Zn, Zn + Fe, and Fe applied to growth media at sowing. N1 and N2 for single and split N. 80, 120, and 160 stand for rate of N in kg ha−<sup>1</sup> . Bars with same alphabet at head are not significantly different at 5% level of significance. ∗∗Significant at p = 0.01; #Not significant at p = 0.05; Avg., Average; Treatment means ± 1 SE (n = 4) followed by same upper case alphabet are not significantly different for N- × Zn–Fe-treatments. Average means followed by same lower case alphabet are not significantly different for respective N- or Zn–Fe- treatments. Tukey comparison was performed at 5% level of significance. N1 and N2 stand for single and split application of N. N120 stands for growth media application of 120 kg N ha−<sup>1</sup> . Zn, Zn + Fe and Fe without suffix for growth media application of Zn–Fe- treatments and with suffix's + f' for growth media plus foliar application. TABLE 4 \| Mean ± 1 SE (n = 4) whole grain protein (%) in wheat grains at experiment with growth media plus foliar application of Zn and Fe. ∗∗∗Significant at p = 0.001; <sup>∗</sup> significant at p = 0.05; Avg., Average. The significance of letters after means and treatments are explained in Table 3. of grains pot−<sup>1</sup> and grain Fe-uptake (Equation 1) rather than with a single variable. The coefficient of TGW and number of grains pot−<sup>1</sup> were negative, but positive for grain Fe-uptake. It indicated that grain Fe-concentration had a tendency to increase when total Fe-uptake in the grain increased but tend to lower with increase in grain yield parameters: TGW and number of grains pot−<sup>1</sup> . Grain Fe − concentration (mg kg−<sup>1</sup> ) = 69.89 − 0.96 TGW (g) − 0.321 No. of grains pot−<sup>1</sup> + 261.46 Grain Fe − uptake grain (mg pot−<sup>1</sup> ) (1) p < 0.001 (for regression model, No. of grains pot−<sup>1</sup> , Grain Fe- uptake); p > 0.05 for TGW; Similarly, a regression analysis of Zn concentration in grain with TGW, number of grains pot−<sup>1</sup> and grain Feuptake in together showed a significant relation (p < 0.01) (Equation 2). The regression model defined about 72% of the variability in the grain Zn- concentration indicating the role of other variables in its determination. Positive coefficients of TGW and total Zn- uptake in grain indicated that grain Znconcentration tend to increase with these factors and negative coefficient for number of grains pot−<sup>1</sup> hint for decrease in FIGURE 2 \| Mean ± 1 SE (n = 4) bar plot for the responses of grain Fe- concentration at N-treatments when Zn + Fe and Fe applied to growth media at sowing. N1 and N2 for single and split N. 80, 120, and 160 stand for rate of N in kg ha−<sup>1</sup> . Bars with same alphabet at head are not significantly different at 5% level of significance. alphabet at head are not significantly different at 5% level of significance. grain Zn- concentration when number of grains pot−<sup>1</sup> tended to increase. Grain Zn − concentration (mg kg−<sup>1</sup> ) = 45.625 − 0.646 TGW (g) − 0.211 No. of grains pot−<sup>1</sup> + 120.98 Grain Zn − uptake (content) in grain (mg pot−<sup>1</sup> ) (2) p < 0.001 (for regression model, Grain Zn- uptake; No. of grains pot−<sup>1</sup> ); p > 0.05 (for TGW). ### Localization of Zn and Fe in Wheat Grain Element distribution map of half wheat grains generated by the MA-XRF are shown in Figure 6. Relatively bright spots in the maps represent higher X- ray signal from the respective elements. This signal is influenced by the element concentration in the sample, among other factors. However, the analyzed surfaces of grains were slightly irregular and this gives rise to somewhat hazy depiction of the element distribution. In the MA-XRF maps, it is obvious that Fe and Zn concentrations are rather variable inside the wheat grain. FIGURE 4 \| Mean ± 1 SE (n = 4) bar plot for the responses of grain Zn- concentration at N-treatments when Zn and Zn + Fe applied to growth media at sowing. N1 and N2 for single and split N. 80, 120, and 160 stand for rate of N in kg ha−<sup>1</sup> . Bars with same alphabet at head are not significantly different at 5% level of significance. However, Fe and Zn seem to have similar patterns throughout all grains. Iron concentrated mainly in embryo and to some extent along the aleurone layer in the crease area (Figure 6B). Zn appears to be present in the embryo and along crease just outside the endosperm (Figure 6C). LA-ICP-MS was performed on three out of the six samples that were analyzed by MA-XRF. LA-ICP-MS was used to obtain normalized signals for Fe and Zn along a well-defined line profile transversally across the grains (Figure 7). Generally, the profiles for both elements appear to vary in a similar fashion, confirming their co-localization. The normalized Zn and Fe counts clearly peaked at well-defined points. Higher signals of both elements were found at both ends of the grain and, for Zn only, at the embryo-aleurone interface in the middle of the grain. The (A) Position of wheat grains subjected to MA-XRF. (B–D) Element distribution maps (1400 × 400 pixels) of wheat grains. (B) Iron, (C) Zinc and (D) Potassium. lowest signals for both elements were encountered within the endosperm. Higher normalized signals can be seen for Fe than for Zn at both ends of the grain, generally associated with bran. ## DISCUSSION ### Grain Yield Among N treatments, grain yield increased from the N rate of 80 kg N ha−<sup>1</sup> (N1N80) to the N rate of 120 kg N ha−<sup>1</sup> (Ntreatments: N1N120 and N2N120) but it decreased at the highest N rate of 160 kg N ha−<sup>1</sup> . This suggested positive yield response to increasing N application rate up to a definite rate only (Marino et al., 2009; Abedi et al., 2011) and decline beyond this level. Split application of 160 kg N ha−<sup>1</sup> (70% at sowing and 30% at stem elongation) produced higher grain yield than at split application of 120 kg N ha−<sup>1</sup> . Reduction in grain yield at higher N rate may be associated to dilution of Zn and Fe, thus limiting their supply. As pointed out by Kutman et al. (2011), significant reduction in grain yield was caused by the high N treatment under the discontinued Zn regime. Dilution of Zn may have affected grain yield by impairing the reproductive development (Cakmak and Engels, 1999). It is likely that the reproductive development in later spikes was negatively affected by poor supply of Zn (Kutman et al., 2011). Grain yield was significantly higher for growth media application of Zn than growth media applied Fe or Fe + Zn. The results are in line with Silspour (2007) and Nadim et al. (2012). Nadim et al. (2012) recorded significant increase in grain yield with soil applied Zn (in the form of ZnSO4) in comparison with soil applied Fe. The effect of N on grain yield and the increase in grain yield for growth media applied Zn was also associated with the increased number of grain pot−<sup>1</sup> (data not FIGURE 7 \| LA-ICP-MS Normalized counts along scan profiles. Normalized counts represent the relative count intensity of Zn and Fe with respect to K. The signals for Zn and Fe for two scan lines are shown. The arrows indicate approximate location for elevated Zn and Fe signals in the grain fall on scan line. The white part in the picture of the wheat grain is the endosperm and bran is on either sides of endosperm. The embryo is at the top the grain. The bran at right hand side of endosperm is the crease. shown). Kutman et al. (2012) showed that the increase in the grain yield due to improved N and Zn supply was parallel to the increase in spike number and eventually grain amount. Nadim et al. (2012) and Jiang et al. (2013) in their respective studies pointed out higher leaf area index and photosynthetic rate in connection with soil applied Zn at sowing time. Ekiz et al. (1998) noticed significant increase in wheat and other cereals grain yield when Zn (7 kg ha−<sup>1</sup> ) was applied in Zn- deficient soil. ### Whole Grain Protein fpls-09-00307 March 8, 2018 Time: 11:2 # 10 In the experiment with soil application of nutrients, N-treatments N2N160 and N1N80, resulted in comparatively higher WGP than other N treatments. Each of these N- treatments represented different grain yield groups in this study. At N2N160, the grain yield was highest but N1N80 treatment produced lowest yield. At N2N160, the increase in protein with increasing grain yield was supported by increased available N, when 48 kg N ha−<sup>1</sup> was supplied at stem elongation since change in protein with change in yield mainly depends on the available N (Brown et al., 2005). In addition, Brown et al. (2005) illustrated that both increasing and decreasing grain protein with higher grain yield may be due to N surplus firstly and N limitation secondly. Similarly, for N1N80, higher protein was associated with lower yield, suggesting the concentration effect (Marschner, 1995). The higher protein concentration may not be the result of sufficient N but it could be due to the reduction in grain yield by limited available N and environmental limitation (Fowler, 2003) leading to less dilution. Late N application at stem elongation in split N- treatments enhanced grain protein in comparison with single N application at sowing (Elhanis et al., 2000; Abedi et al., 2011). When initial N rate at sowing was sufficient, the split N applied at stem elongation period assured the increase in both yield and protein. For example, split application of 160 kg N ha−<sup>1</sup> (70% at sowing and 30% at stem elongation) increased both protein and grain yield. However, split application of 120 kg N ha−<sup>1</sup> increased protein concentration but not the yield in comparison to single application of 120 kg N ha−<sup>1</sup> at sowing, possibly because of limited availability of initial N needed for an increase of number of grains per spike (Li et al., 2001). ### Iron and Zinc Concentrations in Wheat Grain In general, higher concentrations of Fe and Zn in grain were recorded for the treatments with lower grain yield and lower concentrations when higher grain yields were achieved. Studies in the past have mentioned that dilution of Zn and Fe in wheat grain occurs at increased grain yields (Liu et al., 2006; Gomez-Becerra et al., 2010). Multiple linear regression analysis (Equation 1 and Equation 2) presented a decreasing tendency of Fe- and Zn- concentrations in grain with increase in grain yield parameters: TGW and number of grains pot−<sup>1</sup> . Similarly, Zhao et al. (2009) reported that Zn- concentration of wheat grain correlated negatively with grain yield, but the correlation with grain weight was weak. A positive correlation of Zn- and Fe- uptake (i.e., total uptake in grain) suggested that higher concentrations of Zn and Fe in wheat grain were due to the increased uptake from soil or translocation of Zn and Fe from vegetative parts to the grain (Cakmak et al., 2010; Kutman et al., 2011). A multiple linear regression insinuated a dynamic relation among grain Zn- and Fe- concentrations, their uptake in to grain and grain yield parameters (Marschner, 1995) suggesting that the process was governed by sink-source relation. The negative coefficients for grain yield components suggested that the dilution of Zn and Fe in grain was due to combined effect of grain size and the number of grains pot−<sup>1</sup> (sink size) as indicated by Sperotto et al. (2013) in rice plant, pointing involvement of factors other than grain (sink strength) only. Other factors could be the availability of metals (Marschner, 1995), for instance, Zn and Fe during grain filling (Cakmak et al., 2004, 2010; Kutman et al., 2011) or factors contributing dry weight (starch) in grain, which increases the size, and weight of grain (Marschner, 1995; Pleijel et al., 1999). The application of 30% higher Zn and Fe, either separately (Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> and Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ) or together (Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ), as foliar spray in addition to soil application caused positively significant increase in Zn- (Kutman et al., 2010) and Fe-concentrations in grain (Cakmak et al., 2010; Habib, 2012). For instance, foliar applied Fe (F<sup>s</sup> <sup>+</sup> <sup>f</sup> ) and Zn + Fe (Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ) increased the Fe- concentration in grain by 34 and 64% in comparison to growth media applied Fe and Zn + Fe, respectively. The respective increases for Zn were 17 and 46% for foliar applied Zn (Zn<sup>s</sup> <sup>+</sup> <sup>f</sup> ) and Zn + Fe (Zn + Fe<sup>s</sup> <sup>+</sup> <sup>f</sup> ). This could be explained by the increased activity of Zn and Fe in sources (flag leaf and stem) during grain filling (Cakmak et al., 2010) when additional Zn and Fe was supplied at booting. The increase was notably higher for the application of Zn + Fe together, similar to the finding of Habib (2012), where Fe and Zn concentration in wheat grain increased by applying Zn and Fe together as foliar spray. ### Localization of Zn and Fe in Wheat Grain In this study, concentration map of Zn and Fe, obtained by MA-XRF and normalized count plots provided by LA-ICP-MS, evidenced the co-existence of both elements, especially at embryo stage and just outside the endosperm and the aleurone layer. This is in accordance with the results obtained using staining technique developed by Cakmak et al. (2010), where the colocalization of protein, Zn, and Fe in embryo was claimed to be due to the co-segregation. Similarly, Kutman et al. (2010) also showed the co- existence of Zn and protein in a durum wheat grain. Tsuji et al. (2006) used a µ-XRF technique for the elemental mapping of biological materials and found µ-XRF was useful for the analysis of element distribution in grain samples. In elemental map of black wheat and buck or soba wheat by µ-XRF technique, Zn and Fe were found to be located at either embryo and/or coat of grains (Tsuji et al., 2006). In this study, LA-ICP-MS revealed higher signals for both Zn and Fe in the embryo and bran portions, including the aleurone and crease area, with low signals in the endosperm (Figure 7). These results are similar to those reported by Cakmak et al. (2010) and Wang et al. (2011), where the distribution of Zn in wheat grain and its translocation to the endosperm were shown. Based on the decreasing concentration gradient of Zn from crease area toward endosperm, Cakmak et al. (2010) suggested that Zn and Fe are translocated and distributed through the crease and then pass in to the endosperm. To clearly define the location of Zn and Fe and their gradients, from bran to endosperm, crease area to endosperm and embryo to endosperm, for instance, the identification of the direction of element supply is essential. For this, the spatial resolution should be higher than in this study. Besides, higher sample numbers and improved sample preparation should ensure improved results, allowing for instance to avoid possible topography/surface effects on element signals. ### CONCLUSION The rate of N application at sowing caused an increase in grain and straw yield up to the N rate of 120 kg N ha−<sup>1</sup> and a decrease at higher rate of N. The increase in grain yield was primarily determined by the increase in the number of grains pot−<sup>1</sup> or number of grains spike−<sup>1</sup> . The split application of 160 kg N/ha increased the grain and straw yield more than split application of 120 kg N/ha. The growth media application of Fe and Zn interacted with N to increase protein, Zn and Fe concentration in wheat grain. The foliar sprayed Zn and Fe at booting stage of wheat significantly increased the whole grain protein, total uptake and concentration of Fe and Zn in grain. MA-XRF and LA-ICP-MS results indicated the co-localization of Zn and Fe in grain especially in the embryo and the aleurone. LA-ICP-MS also indicated higher concentration of Zn and Fe in the crease area and lower in the endosperm, indicating that Zn and Fe could translocate into the endosperm (the common source of flour in daily food) via crease tissue. ### REFERENCES ### AUTHOR CONTRIBUTIONS BS: planning of experiment, supervision of student, and writing of the manuscript. YT: conduction of the whole experiment, preparation of samples, and thesis writing for his master degree. OL: assitance in the planning, sample preparation, localization studies, and reading of the manuscript. SC: running of LA-ICPMS and MA-XRF studies and reading of the manuscript. KJ: assistance in LA-ICPMS and MA-XRF studies. ### FUNDING The research part of this master study was financed by the project "Mineral Improved Food and Feed Crops for Human and Animal Health" (Project No. 332160UA) and by a grant from the Norwegian Ministry of Foreign Affairs under the Program for Higher Education, Research and Development (HERD) in Western Balkan. The financial assistance for conducting this study is gratefully acknowledged. We also acknowledge the assistance by CERAD: this study has been funded by the Norwegian Research Council through its Centre of Excellence (CoE) funding scheme (Project No. 223268/F50). This research was supported by the Hercules Foundation (Brussels, Belgium) under grant AUHA09004 and FWO (Brussels, Belgium) Project Nos. G.0C12.13 and G.01769.09. ### ACKNOWLEDGMENTS We wish to thank Matthias Alfeld and Kevin Hellemans for their precious support in data interpretation and measurements by means of MA-XRF and LA-ICPMS, respectively. This manuscript is based on Mr. Timsina's thesis and that this thesis is the only medium this content has appeared in and that the publication of this content is in line with the policy of the Norwegian University of Life Sciences. Brown, B., Westcott, M., Christensen, N., Pan, B., and Starck, J. (2005). Nitrogen Management for Hard Wheat Protein Enhancement. Oregon, DC: PNW, 578. Ethylenediaminetetraacetic acid. J. Agric. Food Chem. 56, 4643–4649. doi: 10.1021/jf800041b Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Singh, Timsina, Lind, Cagno and Janssens. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis #### David Chan-Rodriguez1,2 and Elsbeth L. Walker<sup>2</sup> \* <sup>1</sup> Plant Biology Graduate Program, University of Massachusetts Amherst, Amherst, MA, United States, <sup>2</sup> Department of Biology, University of Massachusetts Amherst, Amherst, MA, United States The micronutrient iron (Fe) is essential for photosynthesis, respiration, and many other processes, but it is only sparingly soluble in aqueous solution, making adequate acquisition by plants a serious challenge. Fe is a limiting factor for plant growth on approximately 30% of the world's arable lands. Moreover, Fe deficiency in humans is a global health issue, affecting 1.62 billion people, or about 25% of the world's population. It is imperative that we gain a better understanding of the mechanisms that plants use to regulate iron homeostasis, since these will be important targets for future biofortification and crop improvement strategies. Grasses and non-grasses have evolved independent mechanisms for primary iron uptake from the soil. The grasses, which include most of the world's staple grains, have evolved a distinct 'chelation' mechanism to acquire iron from the soil. Strong iron chelators called phytosiderophores (PSs) are synthesized by grasses and secreted into the rhizosphere where they bind and solubilize Fe(III). The Fe(III)-PS complex is then taken up into root cells via transporters specific for the Fe(III)- PS complex. In this study, 31 novel, uncharacterized striped maize mutants available through the Maize Genetics Cooperation Stock Center (MGCSC) were analyzed to determine whether their mutant phenotypes are caused by decreased iron. Many of these proved to be either pale yellow or white striped mutants. Complementation tests were performed by crossing the MGCSC mutants to ys1 and ys3 reference mutants. This allowed assignment of 10 ys1 alleles and 4 ys3 alleles among the novel mutants. In addition, four ys<sup>∗</sup> mutant lines were identified that are not allelic to either ys1 or ys3. Three of these were characterized as being non-allelic to each other and as having low iron in leaves. These represent new genes involved in iron acquisition by maize, and future cloning of these genes may reveal novel aspects of the grass iron acquisition mechanism. Keywords: iron, phytosiderophores, yellow stripe, maize, mutants ### INTRODUCTION The global demand for crops with high concentrations of nutrients in edible tissues is increasing due to current trends in population growth, global climate change, and decreasing arable land resources (Eckardt et al., 2009). Iron (Fe) deficiency in humans is a global health issue, affecting 1.62 billion people, or about 25% of the world's population, and it is imperative that we gain a better ### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil ### Reviewed by: Yoshiko Murata, Suntory Foundation for Life Sciences, Japan Sebastien Thomine, Centre National de la Recherche Scientifique (CNRS), France > \Correspondence: Elsbeth L. Walker [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 01 December 2017 Accepted: 29 January 2018 Published: 20 February 2018 #### Citation: Chan-Rodriguez D and Walker EL (2018) Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis. Front. Plant Sci. 9:157. doi: 10.3389/fpls.2018.00157 understanding of the mechanisms that plants use to regulate iron homeostasis, since these will be important targets for future biofortification strategies (McLean et al., 2009; Murgia et al., 2012). Quantitative trait loci (QTL) have been identified for maize grain iron accumulation (Zhang et al., 2017) and identification of additional components of the maize iron homeostatic apparatus may help to elucidate the genes underlying such QTL. Although we have learned a great deal through the study of model organisms such as Arabidopsis, it is important to note that the grasses, which include most of the world's staple grains, use phytosiderophores (PSs) that are secreted into the rhizosphere where they bind and solubilize Fe(III) (Tagaki, 1976; Tagaki et al., 1984, 1988). PSs are not made or used by non-grass species. Biofortification of crops has been restricted by our limited knowledge of the molecular mechanisms controlling iron uptake, translocation, accumulation, and deposition in the grain. Attempts to increase iron content have been promising, but these efforts have been focused on the relatively small set of known genes that are involved in iron homeostasis. The iron-storage protein, ferritin (Briat and Lobreaux, 1998; Briat et al., 1999), has been expressed in rice endosperm, to increase iron and zinc content (Goto et al., 1999; Drakakaki et al., 2005). The iron uptake machinery has been a target for biofortification by engineering key enzymes involved in PS synthesis (Higuchi et al., 1999; Takahashi et al., 1999, 2001; Suzuki et al., 2008). These efforts have been only partially successful, suggesting that identifying additional genes involved in mobilization and translocation within the plant could be helpful to develop additional strategies for the production of biofortified crops. In plants, iron is essential for photosynthesis, respiration, and many other processes, but is only sparingly soluble in aqueous solution, making adequate acquisition by plants a serious challenge (Marschner, 1995). Furthermore, iron is highly reactive and if over-accumulated can cause cellular damage. As a response to these key properties of iron, plants have evolved highly regulated iron mechanisms to ensure efficient and tightly controlled acquisition from the soil. Most plants use a combination of rhizosphere acidification, iron reduction, and uptake via the ZIP (ZRT, IRT-like protein) family transporter, IRT1 (iron-regulated transporter). In this strategy, iron is first solubilized and then taken up from the soil, as reviewed in Walker and Connolly (2008), Jeong and Guerinot (2009), and Morrissey and Guerinot (2009). In contrast, the grasses, which include most of the world's staple grains, have evolved a distinct 'chelation' mechanism to acquire iron from the soil. PSs are synthesized by grasses and secreted into the rhizosphere where they bind and solubilize Fe(III) (Tagaki, 1976; Tagaki et al., 1984, 1988). The Fe(III)-PS complex is then taken up into root cells via transporters specific for the Fe(III)-PS complex (Romheld and Marschner, 1986; von Wiren et al., 1994). This mechanism is also known as 'Strategy II.' The Fe(III)-PS uptake transporter Yellow Stripe1 (YS1) has been studied extensively (Curie et al., 2001; Yen et al., 2001; Roberts et al., 2004; Schaaf et al., 2004; Murata et al., 2006; Harada et al., 2007; Inoue et al., 2009; Lee et al., 2009), and is a proton-coupled symporter of Fe(III)-PS complexes (Schaaf et al., 2004). Phytosiderophores are chemically quite distinct from bacterial and fungal siderophores (Miethke and Marahiel, 2007) and belong to a class of compounds called mugineic acids (Ma and Nomoto, 1996), with a well-worked out biosynthesis (Mori and Nishizawa, 1987; Kawai et al., 1988; Shojima et al., 1990; Ma et al., 1995; Takahashi et al., 1999; Kobayashi et al., 2001). In contrast to the details established for PS biosynthesis and Fe-PS uptake, the molecular details of PS secretion have not been as well-characterized. In several grass species, PSs are secreted according to a diurnal cycle, with release occurring several hours after sunrise (Zhang et al., 1991; Walter et al., 1995; Ma et al., 2003; Reichman and Parker, 2007; Ueno et al., 2007; Nagasaka et al., 2009; Bernards et al., 2014). Large numbers of vesicles have been observed in barley roots just prior to the daily release of PS, suggesting that PSs are secreted by exocytosis (Nishizawa and Mori, 1987; Sakaguchi et al., 1999; Negishi et al., 2002). Furthermore, microarray analysis of barley roots indicated that expression of genes associated with polar vesicle transport increases in the early morning (Negishi et al., 2002). The anion channel blockers anthracene-9-carboxylic acid and phenylglyoxal were shown to inhibit PS secretion by barley roots (Sakaguchi et al., 1999), potentially indicating that anion channels are involved in loading PS to secretory vesicles. Alternatively, anion channels in the plasma membrane (PM) could be responsible for PS transport across the PM. Major facilitator superfamily transporters with PS efflux activity were recently identified in rice and barley, and have been called transporter of mugineic acid (TOM1) (Nozoye et al., 2011). A classically known mutation in maize called yellow stripe3 (ys3; Wright, 1961) renders plants unable to secrete PSs, even though PSs are synthesized in normal amounts (Basso et al., 1994; Lanfranchi et al., 2002). The Ys3 gene in maize is located between 85,618,053 and 114,789,459 on chromosome 3 based on two genetic markers (IDP3861 and IDP4688) on the IBM2 2008 Neighbors map. A partial gene with similarity to TOM1 (GRMZM2G063306 also called ZEAMMB73\_058478) is located within this interval in the maize reference sequence version 3 but the sequence contained two sequence gaps in the region occupied by GRMZM2G063306. Based on sequence similarity and strong expression during iron deficiency, this gene was suggested as a candidate for the locus affected in ys3 mutants (Nozoye et al., 2013; Li et al., 2014), but genetic evidence for this assignment has not been presented. In spite of this progress in understanding the process of PS synthesis, release, and uptake of Fe-PS complexes, there are many gaps in our understanding of what makes a particular grass species or cultivar 'iron efficient.' In Kentucky bluegrass, for example, the amount of PS release does not correlate well with resistance to iron deficiency (Buxton et al., 2012). Because of this complexity, we sought to understand the genes in Zea mays (maize) that contribute to iron efficiency, by examining the set of maize mutants available through the Maize Genetics Cooperation Stock Center (MGCSC) that have been described as 'yellow striped' or 'green striped.' Both these descriptions may refer to iron deficiency chlorosis that is typical in both ys1 and ys3 maize mutants, and is characterized by yellow interveinal regions and green veins. By performing allelism tests with ys1 and ys3 reference mutant plants, we have identified novel yellow striped mutants (that we designate as ys<sup>∗</sup> , pending gene identification and assignment of new nomenclature) that may shed light on additional components contributing to iron efficiency in maize. We further characterized the sequence of GRMZM2G063306 in the WT B73 genome and the ys3 reference mutants. We have identified four new alleles of ys3. Based on the evidence from our sequencing of GRMZM2G063306 in multiple independent ys3 mutants, we present strong genetic evidence that GRMZM2G063306 (ZmTOM1) corresponds to the Ys3 gene of maize. ### MATERIALS AND METHODS ### Plant Material and Growth Conditions Maize (Zea mays) plants of B73 and W22 inbred lines were used as WT reference in our experiments, as indicated in the text. Uncharacterized yellow striped mutants were obtained from the MGCSC<sup>1</sup> . For all experiments involving genetic crosses, stocks were grown at the University of Massachusetts Crop and Animal Research and Education Center, South Deerfield, MA, United States, during the summer season between May and September. Mutant plants were supplemented with foliar iron (Fe-EDDHA) through growing season to alleviate chlorosis. For the purposes of initial phenotyping, plants were grown in the greenhouse in a 4:1 v/v mix of potting soil and Turface. All phenotyping was also repeated under field conditions. Supplemental light was supplied with high-pressure sodium lamps to give a 20 h light period each day. For quantitative polymerase-chain-reaction (PCR) analysis, plants were grown in a sand:Turface mix (9:1 v/v) irrigated with water until germination and then irrigated with modified Hoagland's nutrient solutions, with 1 mM KH2PO4, 3.75 mM KOAc, 5 mM Ca(NO3)2, 1.25 mM KNO3, 2 mM MgSO4, 3.75 mM NH4OAc, 46 uM H3BO3, 9.1 uM MnCl2, 0.77 uM ZnSO4, 0.32 uM CuSO4, and 0.83 uM H2MoO<sup>4</sup> (Yordem et al., 2011) containing 100 µM FeSO4-EDTA every 48 h. Plants were grown for 10 days after germination before the root tissue was collected. ### PCR and Sequencing of ZmTOM1 in ys3 Mutant Lines Genomic DNA was extracted from leaves of ys3 mutant plants (ys3:04HI-A632GN-144, ys3:67-2403, ys3:04HI-Oh43xA632GN-187, and ys3:07IL-B73GN-279) and the exons of ZmTOM1 (GRMZM2G063306/Zm00001d041111) were amplified using primers listed in Supplementary Table 1. Amplifications were performed using ExTaq polymerase (Takara, Madison, WI, United States), with cycling conditions of 95◦C, 60 s followed by 35 cycles of 95◦C, 15 s, 55◦C, 30 s, and 72◦C, 60 s, with a final extension step at 72◦C for 5 min. PCR products were gel purified before sequencing. ### Real-Time PCR Analysis (qRT-PCR) The root tissue was flash frozen in liquid nitrogen after harvesting. The frozen root tissue was ground using a Tissuelyser (QIAGEN, Valencia, CA, United States) in 2 ml tubes containing 3.2 mm chrome steel beads (BioSpect Products, Bartlesville, OK, United States). Total RNA was extracted using QIAGENR <sup>R</sup> Neasy Plant Mini Kit (QIAGEN, Valencia, CA, United States), and on-column DNAse treatment step was included for all samples. cDNA was synthesized from 750 ng of total RNA using SuperScript IV VILO (Life Technologies, Carlsbad, CA, United States). For real-time PCR (RT-PCR) analysis, Quantprime primer design webtool (Arvidsson et al., 2008) was used to design ZmTOM1 primers. The primer efficiency of each set of primers (Supplementary Table 2) was evaluated empirically by serial dilution curve of cDNA. PowerUPTM SYBRTMGreen Master Mix (Life Technologies, Carlsbad, CA, United States) was used in quantitative RT-PCR experiments. A two-step PCR protocol was used with the following conditions: initial cycle of 50◦C, 120 s, and 95◦C, 120 s, and 40 cycles of 95◦C, 15 s, and 60◦C, 60 s. After two-step cycling was completed, melting curve was performed to ensure that single amplicon was obtained from each reaction. To determine transcript levels, the threshold cycle (Ct) values from target gene was normalized to ZmGAPDH reference gene for each sample and by the 11C<sup>t</sup> method, we calculated fold change compared to B73 WT. Data represent three biological replicates. ### Inverse PCR Genomic DNA (∼1 µg) was digested with AciI and NlaIII (New England Biolabs) for 2.5 h at 37◦C and reaction was stopped by incubating for 20 min at 65◦C. The DNA was then diluted 25 fold, and ligation was performed using 20 units of Epicenter <sup>R</sup> T4 DNA ligase (Illumina, Inc., Madison, WI, United States) overnight at either 20◦C for blunt ends or 4◦C for sticky ends. The resulting ligation was purified using phenol/chloroform (1:1, v/v) and ethanol precipitation in the presence of 40 µg of glycogen. Then, 1/6 of the purified ligation was used as template for the 1st round of PCR, with primers oZmTOM1\_4456 and oZmTOM1\_4504 for AciI restriction digest, or primers oZmTOM1\_3641 and oZmTOM1\_5012 for NlaIII restriction digestions. The 2nd round of PCR was performed using 1 ul of a 1:100 dilution of the PCR product from the 1st round as template using nested primers oZmTOM1\_4338 and oZmTOM1\_4573for AciI digested DNA or oZmTOM1 4774 and oZmTom1\_5154, for NlaIII digested DNA. Amplifications were performed using ExTaq polymerase (Takara, Madison, WI, United States), with cycling conditions of 95◦C, 2 min, and 25 cycles of 95◦C, 15 s, 57◦C, 30 s, 72◦C for 2 min, and a final elongation step for 10 min. Primer sequences for inverse PCR (iPCR) are listed in Supplementary Table 3. ### Metal Measurement Leaves of at least 10 individual plants were collected from 19 day-old plants grown in the greenhouse and samples were dried at 65◦C for 72 h. In every experiment, all controls and mutants were grown simultaneously and using the same soil batch. Metal <sup>1</sup>http://maizecoop.cropsci.uiuc.edu/ concentrations were determined by inductively couple plasma mass spectrometry (ICP-MS) at the Donald Danforth Plant Research Institute. ### RESULTS ### Complementation Testing of Yellow Striped Mutants from MGCSC We obtained 31 mutants classified as having a yellow striped phenotype from the MGCSC (Table 1). These were planted in the field and phenotypic analysis indicated that 21 of the lines showed the phenotype typical of iron deficiency chlorosis. In the other 10 lines, we either did not observed stripes at all or else observed a solid yellow or white striped phenotype (Table 1). To identify new genes involved in iron uptake in maize, and to identify new alleles for ys3, we performed complementation tests between the uncharacterized yellow striped mutants and the reference maize mutants ys1:ref and ys3:ref. Due to stunting or sterility of some mutant stocks, not all crosses were obtained. From these crosses, we identified 10 new alleles for ys1 and 4 new alleles for ys3. Moreover, we found four novel yellow stripe mutants, ys<sup>∗</sup> -PI262172, ys<sup>∗</sup> :N2398, ys<sup>∗</sup> :PI228180, and ys<sup>∗</sup> :04HI-A632XOh43GN-18, that are not allelic to ys1 or ys3, and thus may represent new maize genes involved in iron uptake or homeostasis. TABLE 1 \| Yellow or green striped mutants from the Maize Genetics Cooperation Stock Center (MGCSC) and results of complementation tests with ys3:ref and ys1:ref. NT indicates that allelism was not tested. sequence is shown for comparison. For ys3:04HI-Oh43XA632-GN-187, genomic sequence of the site of insertion is shown, with the 8 bp direct repeat flanking the ### Analysis of ZmTOM1 Coding Sequence in ys3 Alleles Because a gene with similarity to TOM1 (GRMZM2G063306 also called ZEAMMB73\_058478, here designated ZmTOM1) is located within the genomic interval containing Ys3, this gene has been suggested as a candidate for the locus affected in ys3 mutants (Nozoye et al., 2013; Li et al., 2014). To determine whether the suggested candidate gene, ZmTOM1, underlies the long-known ys3 mutant, we sequenced the exons of ZmTOM1 in all five alleles of ys3 (reference allele and the four novel alleles identified through complementation tests; Figure 1) to identify causative mutations. The MGCSC holds three stocks designated as ys3:ref mutants (304A, 311F, and 311G). In all three lines, ZmTOM1 sequences were identical, and contained a 4 bp insertion in exon 9. This insertion causes a frame shift followed by 11 novel amino acids before introducing a premature stop codon (Figure 1). We found a different 4 bp insertion in exon 11 of ZmTOM1 in the ys3:67-2403 allele (Figure 1). This 4 bp insertion causes a frame shift followed by 107 new amino acids before a stop codon occurs to terminate the protein prematurely. For the ys3:04HI-Oh43XA632-GN-187 allele, we could not amplify fragments containing exon 10 and 11, but partial sequences from both exons could be obtained. We hypothesized that an insertion could be present between these two exons causing failure to amplify that region. Using iPCR, we identified both left and right borders of an insertion containing the characteristic elements of a transposon. The inserted sequences were flanked by 8 bp direct repeats and contained 130 bp terminal inverted repeats (TIRs). We aligned the TIR sequences with the maize reference sequence and identified two regions in chromosome 7 annotated as Far1-related sequence 5, which corresponds to a mutator-like transposable element (MULE). MULE transposons insertion underlined. generate 8–10 bp target sequence duplications and have TIRs of >100 bp. Thus, the insertion has all the elements expected for a MULE transposon inserted in the ys3:04HI-Oh43XA632- GN-187 allele (Figure 1). A one nucleotide change at the exon– intron border for exon 5 of ys3:04HI-A632GN-144 was observed. Likewise, a one nucleotide change near the 3<sup>0</sup> end of exon 9 was observed in ys3:07IL-B73GN-279. We hypothesized that splicing could be affected these two alleles, and so investigated ZmTOM1 gene expression in the roots of these plants using Q-RT-PCR. Expression of ZmTOM1 was observed in both ys3:04HI-A632GN-144 and ys3:07IL-B73GN-279 (data not shown). Since the ZmTOM1 transcript was observed, we speculated that altered splicing due to the mutations might be leading to aberrant ZmTOM1 mRNA, so we sequenced the full-length cDNA from each mutant line to test this. We confirmed that the one nucleotide change at the ys3:04HI-A632GN-144 exon–intron junction altered the splice donor site. The mutation causes splicing to occur at a new donor site 3 nucleotides into the adjacent intron (Figure 2). As a result, one additional amino acid is inserted without affecting the reading frame. The amino acid is inserted in a strongly conserved region that could lead to a nonfunctional protein. In the ys3:07IL-B73GN-279 allele, the single nucleotide change occurred at the first nucleotide of intron 9, changing the splice donor site from GT to AT. In the mRNA produced by this allele, a new splice donor site is recognized in exon 9, 21 nucleotides upstream from the original donor site (Figure 2). The resulting amino acid sequence is thus missing seven residues in a strongly conserved region. Our results show clear genetic evidence that the Ys3 gene is ZmTOM1. ### Analysis of Novel Yellow Striped Maize Mutants To evaluate whether the yellow striped phenotype in ys<sup>∗</sup> mutants is due to low iron, we analyzed metal levels in leaves of three of the mutants. The ys<sup>∗</sup> -PI262172 mutant was not included in this analysis, because its stunted growth prevented our obtaining sufficient material for this experiment. Visual inspection of the leaves of 12-day-old WT and mutant plants indicates differences in the severity of the observed chlorosis, with ys<sup>∗</sup> :PI228180 having very mild chlorosis and ys∗ :04HI-A632xOh43GN-18, ys1:ref, and ys3:ref having the most marked chlorosis (Figure 3). In all three ys<sup>∗</sup> mutants tested, the levels of iron were significantly lower than WT (Figure 3) indicating that the plants are iron-deficient. Control ys1 and ys3 plants are also low in iron, as expected. In a segregating population of ys<sup>∗</sup> :04-04HI-A632xOh43GN-18 mutants, the iron concentration in yellow striped siblings was less than half (42%) that of WT siblings. The iron concentration in ys<sup>∗</sup> :04HI-A632xOh43GN-18 was significantly lower even than ys1 and ys3, indicating a very substantial alteration in iron homeostasis in these plants. For ys<sup>∗</sup> :PI228180 and ys<sup>∗</sup> :N2398, iron levels were higher than either ys1 or ys3, TABLE 2 \| Complementation test results among ys<sup>∗</sup> mutants. but were still significantly lower than the amount in WT control plants. We also measured the Zn and Mn concentration in the leaves of the mutant plants (Figure 3). We note that altered iron homeostasis often causes alterations to multiple metals. For example, ys1 and ys3 mutants, which are clearly impaired in iron uptake, have higher Zn and Mn than WT control plants (Figure 3). It is possible that this occurs because PS secretion or uptake directly affects Mn and Zn uptake or translocation, but it is also possible that the mechanism is indirect. Like ys1 and ys3 mutants, ys∗ :04HI-A632xOh43GN-18 and ys<sup>∗</sup> :N2398 plants have higher Mn and Zn than WT control plants. For ys<sup>∗</sup> :PI228180, the Zn concentration in leaves is not significantly different from WT control plants, and the Mn concentration is slightly but significantly lower than that of the WT controls, and much lower that the Mn concentration in the ys1 and ys3 mutants. We note that the soil batch used for growth of the ys<sup>∗</sup> :04HI-A632xOh43GN-18 and ys<sup>∗</sup> :N2398 plants and their controls was different from the batch used to grow ys<sup>∗</sup> :PI228180 and its controls. ### Complementation Tests among ys<sup>∗</sup> Mutants We performed crosses among three of the four identified ys<sup>∗</sup> mutants to determine how many loci are represented by these three mutants. The ys<sup>∗</sup> -PI262172 mutant was not included in this analysis, because its stunted growth prevented our obtaining the appropriate crosses. F1 seeds were grown in the greenhouse and the phenotypes were recorded. We found complementation among all crosses performed in ys<sup>∗</sup> mutants, indicating that they do not represent alleles. These results show that we have identified three novel genes involved in iron homeostasis (Table 2). ## DISCUSSION ### The Rationale for Gene Discovery in Zea mays Much of the molecular work on iron uptake and homeostasis in grasses has been performed using rice, both because of its properties as a model organism and also because of the fundamental importance of this species as a crop. Still, the Fe(III)-PS uptake transporter, YS1, was first identified in maize by making use of the excellent genetic resources available in this species (Curie et al., 2001). The YS1 gene has been used directly as a strategy for engineering biofortification with mixed results. In an early study using constitutive expression of barley YS1 in rice, plants showed superior growth in alkaline soil conditions but did not contain significantly more iron in grains (Gomez-Galera et al., 2012). Later, barley YS1 expressed in rice was shown to promote the preferential mobilization and loading of Fe in seeds while displacing Cd and Cu (Banakar et al., 2017). At present, two key uptake genes, Ys1 and Ys3 (TOM1), for the grass specific mechanism are understood, as are the genes involved in PS synthesis, but it is unclear whether additional grass specific components exist. If they do, they will need to be discovered directly in grass species such as maize. ### Identification of the Ys3 Gene Maize ys3 mutants lack the ability to secrete PS (Lanfranchi et al., 2002). ZmTOM1 has been proposed as candidate gene for Ys3 because of its function as PS effluxer (Nozoye et al., 2011) and its location within the same map interval as the genetically identified ys3 mutant allele. Previous reports analyzing the ys3 transcriptome during iron deficiency suggested reduced expression and alternative splicing of ZmTOM1 (Nozoye et al., 2013). However, this approach could not definitively assign ZmTOM1 as the Ys3 gene, since other genes (ZmMATE3/ZmPEZ1) had reduced expression in ys3 mutants, and mutations in ZmTOM1 were not identified. Here, we were able to show that multiple alleles of ys3 could be found among the yellow striped mutants held at the MGCSC, and that each of these carries a unique mutation that is expected to abolish the function of the ZmTOM1 protein. ### Three Novel Yellow Striped Maize Mutants In this study, we identified three novel yellow striped mutants whose phenotype is apparently caused by low iron content. These mutants represent three different loci involved in iron ### REFERENCES homeostasis. Genetic mapping to identify the underlying genes responsible for the yellow striped phenotype in these mutants will reveal unknown elements of the iron homeostasis machinery and may provide new options for biofortification. Initially, it appeared as though our screening of the MGCSC mutant collection had reached saturation since multiple alleles for both ys1 and ys3 were obtained. However, three novel loci contributing to iron content in leaves were identified, indicating that saturation mutagenesis has likely not been reached and additional genes causing an iron deficiency induced yellow striped phenotypes in maize could be uncovered. Genetic mapping of the three ys<sup>∗</sup> mutants is underway to discover the genes responsible for these interesting metal homeostasis phenotypes. Future work will also include tests to indicate whether additional iron supply or direct iron supply to the leaves can alleviate the ys<sup>∗</sup> phenotypes, and tests of the iron concentration in grains of the mutant plants to see whether the grain concentration of iron is altered in the mutants. ### AUTHOR CONTRIBUTIONS EW conceived the project, was responsible for the experimental design, and also performed some of the genetics crosses and phenotyping in the field. DC-R conducted screening and genetics crosses and performed all of the molecular work on the project. ### FUNDING This work was supported by grants to EW (USDA AFRI Grant Nos. 2009-02268 and NSF IOS-153980). ## ACKNOWLEDGMENTS We would like to express our sincere gratitude to the lab members, past and present who helped in the cornfield, especially Rakesh K. Kumar and Harry Klein, to Mary Sachs, of the MGCSC, who answered numerous questions and provided us with details pertaining to the stocks used in these studies, and to Dan Jones and Chris Phillips for their expert assistance in the greenhouse. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00157/ full#supplementary-material metals by selective Fe transport. Plant Biotechnol. J. 15, 423–432. doi: 10.1111/ pbi.12637 Wright, J. (1961). A new yellow stripe on chromosome 3. Maize Newslett. 35:111. Conflict of Interest Statement:* The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Chan-Rodriguez and Walker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Contribution of NtZIP1-Like to the Regulation of Zn Homeostasis Anna Papierniak<sup>1</sup> , Katarzyna Kozak<sup>1</sup> , Maria Kendziorek<sup>1</sup> , Anna Barabasz<sup>1</sup> , Małgorzata Palusinska ´ 1 , Jerzy Tiuryn<sup>2</sup> , Bohdan Paterczyk<sup>3</sup> , Lorraine E. Williams<sup>4</sup> and Danuta M. Antosiewicz<sup>1</sup> \* 1 Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland, <sup>2</sup> Faculty of Mathematics, Informatics, and Mechanics, University of Warsaw, Warsaw, Poland, <sup>3</sup> Laboratory of Electron and Confocal Microscopy, Faculty of Biology, University of Warsaw, Warsaw, Poland, <sup>4</sup> Biological Sciences, University of Southampton, Southampton, United Kingdom Tobacco has frequently been suggested as a candidate plant species for use in phytoremediation of metal contaminated soil but knowledge on the regulation of its metal-homeostasis is still in the infancy. To identify new tobacco metal transport genes that are involved in Zn homeostasis a bioinformatics study using the tobacco genome information together with expression analysis was performed. Ten new tobacco metal transport genes from the ZIP, NRAMP, MTP, and MRP/ABCC families were identified with expression levels in leaves that were modified by exposure to Zn excess. Following exposure to high Zn there was upregulation of NtZIP11-like, NtNRAMP3, three isoforms of NtMTP2, three MRP/ABCC genes (NtMRP5-like, NtMRP10-like, and NtMRP14 like) and downregulation of NtZIP1-like and NtZIP4. This suggests their involvement in several processes governing the response to Zn-related stress and in the efficiency of Zn accumulation (uptake, sequestration, and redistribution). Further detailed analysis of NtZIP1-like provided evidence that it is localized at the plasma membrane and is involved in Zn but not Fe and Cd transport. NtZIP1-like is expressed in the roots and shoots, and is regulated developmentally and in a tissue-specific manner. It is highly upregulated by Zn deficiency in the leaves and the root basal region but not in the root apical zone (region of maturation and absorption containing root hairs). Thus NtZIP1-like is unlikely to be responsible for Zn uptake by the root apical region but rather in the uptake by root cells within the already mature basal zone. It is downregulated by Zn excess suggesting it is involved in a mechanism to protect the root and leaf cells from accumulating excess Zn. Keywords: zinc, tobacco, ZIP, NtZIP1-like, yeast complementation ### INTRODUCTION Tobacco (Nicotiana tabacum L cv. Xanthi) has frequently been considered for phytoremediation purposes because of its high biomass and ability to take up and accumulate in leaves high amounts of metals, including zinc (Zn) (Vangronsveld et al., 2009; Herzig et al., 2014; Vera-Estrella et al., 2017). To improve its capacity to take up and store metals in shoots, it has been transformed with a number of metal homeostasis genes, but with limited success (Gisbert et al., 2003; Martínez et al., 2006; Gorinova et al., 2007; Wojas et al., 2008, 2009; Korenkov et al., 2009; Siemianowski et al., 2011; Barabasz et al., 2013; Wang et al., 2015). Recently, it was shown that when expressing metal transporters to engineer new metal-related traits, a major #### Edited by: Raul Antonio Sperotto, University of Taquari Valley, Brazil ### Reviewed by: Manish Kumar Patel, National Institute of Plant Genome Research (NIPGR), India Marc Hanikenne, University of Liège, Belgium > \Correspondence: Danuta M. Antosiewicz [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 01 December 2017 Accepted: 31 January 2018 Published: 16 February 2018 #### Citation: Papierniak A, Kozak K, Kendziorek M, Barabasz A, Palusinska M, Tiuryn J, Paterczyk B, ´ Williams LE and Antosiewicz DM (2018) Contribution of NtZIP1-Like to the Regulation of Zn Homeostasis. Front. Plant Sci. 9:185. doi: 10.3389/fpls.2018.00185 264* part of the resulting phenotype was due to the modulation of endogenous gene expression (Barabasz et al., 2016; Kendziorek et al., 2016). Therefore, a greater understanding of Zn-homeostasis mechanisms is required to successfully genetically modify the efficiency of Zn accumulation in shoots. Maintaining high Zn in the above ground organs depends on three major processes operating efficiently: Zn uptake from the soil, root-to-shoot translocation and storage in leaves without detrimental toxic effects. Zn uptake is thought to be mediated primarily by ZIP (ZRT\IRT related Protein) metal transporters. In Arabidopsis thaliana AtZIP2, AtIRT1 and AtIRT3 residing in the plasma membrane have been identified as key players in Zn acquisition by roots (Vert et al., 2002; Lin et al., 2009; Palmer and Guerinot, 2009; Milner et al., 2013). The root-to-shoot translocation of Zn (and other metals) depends on two main factors: the ability to store the metal in the roots; and the efficiency of its loading into xylem vessels. It has been shown that HMAs (Heavy-Metal ATPases) which belong to the P1B-ATPase family (Williams et al., 2000; Williams and Mills, 2005) are involved in both processes. HMA3, identified in A. thaliana and rice, localized in the tonoplast of root cortical cells, limits translocation of Cd from the roots to the shoots by sequestrating the metal into the root vacuoles. There is a suggestion that it could also transport Zn into the vacuoles and control the amount of Zn available for xylem loading and thus the efficiency of its translocation to the shoot (Morel et al., 2009; Ueno et al., 2010; Miyadate et al., 2011). The efficiency of the next step in Zn translocation to shoots - loading of a metal into the xylem vessels, is under the control of two genes with overlapping function, HMA2 and HMA4 (Hussain et al., 2004; Verret et al., 2004; Wong and Cobbett, 2009). Encoded proteins are localized in the roots at the plasma membrane of xylem parenchyma cells where they are responsible for Zn (and also Cd) efflux to the xylem. Decreased translocation of Zn to shoots in the athma2athma4 double mutant led to severe Zn deficiency (Hussain et al., 2004; Wong and Cobbett, 2009; Mills et al., 2012). Zn transported to the shoots is stored primarily in the mesophyll cells of leaves. Its level of accumulation depends on the ability of the mesophyll cells to store the metal without toxicity. This complex process involves efficient metal import and its loading into the vacuoles, but also regulated redistribution from this compartment. Currently we are far from having a clear picture of all the elements involved. The potential players include members of several transport families. The ZIP genes play a diverse roles, and those present in the plasma membrane are responsible for Zn uptake, while others localized in the tonoplast could contribute to control of Zn release from vacuoles (Guerinot, 2000; Milner et al., 2013; Ricachenevsky et al., 2015). Accumulation of metal/s in the vacuoles also depends on the NRAMP (Natural Resistance-Associated Macrophage Protein) family. Members of this family transport Fe and Mn, while Cd, Zn or Ni can also serve as substrates for some (Nevo and Nelson, 2006; Ricachenevsky et al., 2015). AtNRAMP1 is a plasma membrane Mn uptake system in roots of A. thaliana; Cailliatte et al. (2010), while NRAMP3 and NRAMP4 are involved in metal release (Mn and Fe) from vacuoles in leaves and seeds (Lanquar et al., 2005, 2010). High expression of NRAMP3 and NRAMP4 genes was noted in the leaves of Zn/Cd hyperaccumulating A. halleri (Weber et al., 2004) and Thlaspi caerulescens. Both TcNRAMP3 and TcNRAMP4 were implicated in metal hypertolerance, but the precise role is yet to be determined (Oomen et al., 2009). Loading of metals into vacuoles is provided by the members of the MTP (Metal Tolerance Proteins) family. Residing in the tonoplast, they are involved in sequestration primarily Zn in the vacuoles, but other metals such as Fe, Mn, Cd, Ni or Co can also be substrates for some family members (Gustin et al., 2011; Menguer et al., 2013; Ricachenevsky et al., 2013; Farthing et al., 2017). However, some MTPs are localized in the plasma membrane, and they remove cations from the cytoplasm to the cell wall (Menguer et al., 2013; Migocka et al., 2015). In the leaves, a key protein for Zn sequestration and detoxification is the vacuolar protein MTP1. AtMTP1 from A thaliana contributes to Zn accumulation in leaves and to basal Zn tolerance by sequestering Zn in vacuoles (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005; Ricachenevsky et al., 2013). MTP1 also has a function in Zn accumulation in shoots of Zn hyperaccumulators such as A. halleri (Dräger et al., 2004) or Thlaspi goesingense (Gustin et al., 2009). Despite a broad interest in the use of tobacco to remove metals from contaminated soil, knowledge of the metal homeostatic processes in this species is still in its infancy. Only a few metal transport genes have been cloned and characterized so far. NtPDR3 (pleiotropic drug resistance) from Nicotiana tabacum was shown to be highly expressed under Fe-deficiency conditions suggesting its involvement in iron homeostasis (Ducos et al., 2005). MTP family members involved in Zn and Co metabolism were cloned from Nicotiana tabacum (NtMTP1a, NtMTP1b) and Nicotiana glauca (NgMTP1) (Shingu et al., 2005). Also, two orthologs of the Arabidopsis thaliana HMA2 and HMA4 were identified in tobacco, NtHMAα and NtHMAβ. Similar to Arabidopsis genes, NtHMAα and NtHMAβ are responsible for Zn and Cd root-to-shoot translocation (Hermand et al., 2014; Liedschulte et al., 2017). Furthermore, studies performed on tobacco BY-2 cells identified two genes encoding Fe uptake proteins; NtNRAMP3 and NtZIP1 (Sano et al., 2012). A second ZIP family member from tobacco, NtIRT1, was also shown to transport Fe, and its expression depended on the level of Fe and Cd in the medium (Yoshihara et al., 2006; Hodoshima et al., 2007). To learn more about the molecular mechanism regulating Zn accumulation in tobacco leaves, the aim of this study was to identify the members of the following key metal transport families that could be involved in regulating Zn levels in the leaf blades: ZIP, NRAMP, and MTP. Moreover, taking into account very limited knowledge on the possible contribution of MRPs (multidrug resistance-associated proteins) family members to detoxification of metals, they were also included. MRP/ABCC (Klein et al., 2006; Verrier et al., 2008) transporters are a ubiquitous subfamily of ABC (ATP Binding Casette) transporters which catalyze the export of substrates out of the cytosol in an ATP-dependent manner. Their involvement in Zn and Cd hypertolerance in N. caerulescens was shown by Halimaa et al. (2014) and also in the detoxification of Cd (Bovet et al., 2003, 2005; Wojas et al., 2007; Gaillard et al., 2008). The major focus in this study was on proteins mediating Zn import into the tobacco leaf cells from the ZIP family. They were identified and initially characterized in several organisms, for example in Arabidopsis (15 ZIPs; Grotz and Guerinot, 2006), rice (16 ZIPs; Chen et al., 2008), bean (23 ZIPs; Astudillo et al., 2013) and more recently, wheat (Evens et al., 2017). In addition to Zn, ZIPs mediate transport of Mn, Fe, Ni, or Cu. Detailed analysis of the role of ZIP genes is still lacking for many of those identified. Their function has been anticipated primarily based on metalspecific (Zn, Fe, Mn, Cd, and Cu) and concentration-dependent (deficit/sufficient/excess) regulation of ZIP expression in organs. (Bashir et al., 2012; Sinclair and Krämer, 2012; Milner et al., 2013; Evens et al., 2017; Nazri et al., 2017). Here, bioinformatics analysis of tobacco genome data was performed to identify sequences homologous to chosen Arabidopsis thaliana metal transport genes, and subsequent expression analysis led to the identification of the NtZIP1-like. It was cloned and characterized indicating its specific function in the regulation of Zn homeostasis in tobacco leaves. ### MATERIALS AND METHODS ### Plant Material and Growth Conditions All experiments were performed on tobacco plants (Nicotiana tabacum var. Xanthi). Surface sterilized seeds (8% sodium hypochloride w/v for 2 min) were germinated on Petri dishes positioned vertically containing quarter-strength Knop's medium, 2% sucrose (w/v) and 1% agar (w/v) (Barabasz et al., 2013). Three weeks following germination, seedlings were transferred to hydroponic conditions. They were cultivated in 2-L pots (5 plants per pot) on aerated quarter-strength Knop's medium for 2 weeks to allow them to adjust to hydroponic conditions. The nutrient solution was renewed every 3–4 days (unless indicated otherwise). Five-week-old plants (3 weeks on plates and 2 weeks on hydroponics) were further used for experiments. They were exposed to chosen Zn (as ZnSO4) concentrations added to quarter-strength Knop's medium. Details are given in the subsections 2.3 and 2.9 below. At the end of each experiment, the plant samples were collected always at the same time of the day (between 10–12 AM). The quarter-strength Knop's medium (containing 0.5 µM Zn) was used as a reference (control) medium in parallel to applied Zn treatments. Plants were cultivated in a growth chamber at temperature 23/16◦C day/night, 40–50% humidity, 16 h photoperiod, and quantum flux density [photosynthetically active radiation (PAR)] 250 mmol m−<sup>2</sup> s −1 , fluorescent Flora tubes. ### Database Search for Putative Tobacco Metal Transport Family Members The goal was to identify potential tobacco metal transporters involved in the accumulation of Zn in leaves. There are two sources of tobacco sequences to be used for gene mining. First, the complete genomic tobacco sequence has recently been made available to the public in GenBank with accession code AWOK00000000 (Sierro et al., 2013, 2014). Second, there is the NCBI database which provides already annotated genes from a range of species including tobacco. In tobacco, only several metal transporters have been already identified, cloned and characterized, some more were annotated and their sequences could be found in the NCBI database. Thus, NCBI database likely does not contain all tobacco genes. Therefore, the search for putative tobacco Zn transporters was performed with the use of both AWOK and NCBI databases. Tobacco metal transporters were identified based on homology to the previously annotated sequences of Arabidopsis thaliana genes belonging to the following major metal transport families: (i) ZIPs: ZRT, IRT-like proteins; (ii) NRAMPs: natural resistance-associated macrophage proteins; (iii) MTP: metal tolerance proteins; (iv) MRP/ABCC: multidrug resistance proteins. The genome of Nicotiana tabacum, Basma Xanthi has been downloaded from http://www.ncbi.nlm.nih.gov/Traces/wgs/?& val=AWOK01 (BX), and a search for tobacco sequences homologous to sequences of metal transporters gene from A. thaliana was performed. For this we used program BLASTn (NCBI Resource Coordinators, 2016) which was run using an amino acid sequence from Arabidopsis against the Basma Xanthi genome. We retained alignments with e-value not exceeding 1e−05. Next we used AAT package (Huang et al., 1997) for analyzing and annotating large genomic sequences containing introns. The predicted exons were further filtered in order to avoid spurious predictions (minimal length at least 10 amino acids, plus a threshold on confidence levels for both boundaries that were returned by AAT package). In parallel, the NCBI database was used for BLASTn searches of Nicotiana sequences with homology to the already annotated A. thaliana sequences of metal transporters. FGENESH and FGENESH+ tools (Softberry, Mount Kisco, NY, United States<sup>1</sup> ) were used to identify the untranslated regions (UTRs), exons, and introns within the scaffold containing sequences of chosen tobacco genes, and to predict putative proteins encoded by these genes. Protein sequence alignments were performed using ClustalW and the phylogenetic trees were constructed with MEGA7.0 software (Tamura et al., 2013) using the maximum likelihood method with 1000 bootstrap replicates. The prediction of membrane-spanning regions and orientation was performed using Phobius software (Käll et al., 2004). ### Identification of Metal Transport Genes Differentially Regulated in Leaves by Exposure to Zn Excess The 5-week old tobacco plants (obtained as described in the section "Plant Material and Growth Conditions") were grown for the next 4 days in the control medium, then they were exposed to 200 µM Zn (added to the quarter-strength Knop's medium) for up to 3 days. Quarter-strength Knop's medium (contains 0.5 µM Zn) served as a reference condition. On the 1st, 2nd, and 3rd day of the Zn treatment blades from the 2nd <sup>1</sup>http://www.softberry.com leaf (counting from the base of a plant) were collected. Leaves were cut out from each plant, petioles and the major midribs were excised, and the fragments of the blades were immediately frozen in liquid nitrogen. Three independent biological replicate experiments were performed. For each repetition, the leaf blade fragments were collected from a total of 40 plants. Quantitative Real-Time PCR (RT-qPCR) was used to determine which putative metal transport genes out of those identified by bioinformatics analysis (see section "Database Search for Putative Tobacco Metal Transport Family Members") are differentially regulated in the leaf blades by 200 µM Zn (as compared with the control conditions). Specific primers were designed for the sequences of identified metal transport genes from the ZIP, NRAMP, MTP, and MRP/ABCC families identified in the tobacco genome databases (Supplementary Table S1). ### Cloning of NtZIP1-Like and Bioinformatic Analysis The whole sequence of ZIP1-like was determined by 5<sup>0</sup> - and 3 0 rapid amplification of cDNA ends (RACE) using SMARTer RACE 5<sup>0</sup> /3<sup>0</sup> Kit (Clontech Laboratories, Inc. and A Takara Bio Company, Mountain View, CA, United States) according to the manufacturer's manual. Briefly, the partial sequence of ZIP1-like (previously identified in the tobacco genome database at AWOK01S302253.1) was used to design genespecific primers (GSPs) for the 5<sup>0</sup> - and 3<sup>0</sup> -RACE reactions [2253- GSP1-1-UPM (5<sup>0</sup> ) 2253-GSP2-1-UPM (3<sup>0</sup> )] (Supplementary Table S1). Amplification of the 5<sup>0</sup> - and 3<sup>0</sup> -end was performed in 50 µl reactions with the use of the Phusion HF polymerase (Thermo Scientific). The PCR product of an expected size was electrophoresed on an 1% agarose/EtBr gel and excised DNA fragment was cleaned with the Macherey-Nagel PCR clean-up Gel extraction (Germany, VWR MANB740609.50) according to the manufacturer's instruction. It was cloned into the pRACE vector (provided with the SMARTer RACE kit) and subsequently the reaction mixture was used to transform Escherichia coli Stellar Competent Cells. The plasmids were isolated from individual colonies, and the presence of the expected insert was confirmed by PCR screening (starters M13/For and M13/Rev), then by sequencing (Genomed, Poland). Nucleic and amino acid sequence alignments between obtained sequence and sequence predicted by Fgenesh program was performed using ClustalW. The full length NtZIP1-like cDNA sequence was amplified by PCR (Supplementary Table S1), subcloned to pENTRTM/D-TOPO <sup>R</sup> and used for E. coli One ShotTM TOP10 (Invitrogen) transformation. The insert was sequenced to confirm the correct sequence. The sequence of the NtZIP1-like cDNA was deposited to the NCBI database (2015) under the accession number XM\_016652513. ### RNA Extraction Total RNA was extracted from samples stored in −80◦C with the use of an RNeasy Plant Kit (Syngen, #SY341010) according to the manufacturer's recommendations, followed by DNase I digestion (Qiagen, #79254). The samples of RNA were quantified at 260 nm using a Nanodrop spectrophotometer ND 100 (Nanodrop, Wilmington, DE, United States). RNA concentration and purity was determined before and after DNA digestion using a NanoDrop spectrophotometer ND-1000 (Nanodrop, Wilmington, DE, United States) and the 260/280-nm ratio showed expected values between 1.8 and 2.0. The RNA integrity of samples was also confirmed by electrophoresis in agarose gel. ### Quantitative Real-Time PCR The cDNA used as a template for the RT-qPCR reaction was synthesized using RevertAidTM First Strand cDNA Synthesis Kits (Fermentas) in a 20 µl reaction volume containing 1–3 µg of aRNA and oligo d(T)18 primers following the manufacturer's protocol. The RT-qPCR reaction was performed according to procedures described in Kendziorek et al. (2016) with minor modifications. It was performed in a Roche mastercycler (LightCycler <sup>R</sup> 480 System, Roche) using Light Cycler480 SYBR Green (Master 0488735001) according to the manufacturer's recommendations. The primers (Supplementary Table S1) were designed using IDT OligoAnalyzer 3.1<sup>2</sup> and OligoCalc: Oligonucleotide Properties Calculator<sup>3</sup> . The tobacco NtPP2A (protein phosphatase 2A; AJ007496) gene was used as the reference gene/internal control and was amplified in parallel with the target gene allowing gene expression normalization and providing quantification. Their stability in the plant samples collected for expression analysis was measured and shown in Supplementary Figure S1. Expression analysis was performed with at least three independent biological replicates. For each sample, reactions were set up in triplicate and means were calculated. Quantification of the relative transcript levels was performed using the comparative dCt (threshold cycle) method. Validation experiments were performed to test the efficiency of the target amplification and the efficiency of the reference amplification. The general quality assessment of the qPCR results was based on the amplification and melting curve profile of the samples in relation to the assay controls (non-template controls). ### Functional Analysis of NtZIP1-Like in Saccharomyces cerevisiae Strains The full cDNA of NtZIP1-like was amplified using Phusion polymerase with the primers introducing XbaI and BamHI restriction sites for amplification (Supplementary Table S1). Obtained sequence was restriction ligated into the pUG35 yeast expression vector (kindly provided by Dr. M. Migocka, The University of Wrocław). The open reading frame (ORF) of NtZIP1-like was inserted in frame C-terminal to the ORF of EGFP (construct pUG35-NtZIP1-like-EGFP) and with the STOP codon (construct pUG35-NtZIP1-like), and fused with the methionine-repressible MET25 promoter (Kurat et al., 2006; Petschnigg et al., 2009). The same cloning strategy has been used to fuse the EGFP coding region to the N-terminal end of the NtZIP1-like in the pUG36 vector (construct pUG36-EGFP-NtZIP1-like). The resulting constructs and empty vectors were transformed to <sup>2</sup>http://eu.idtdna.com/calc/analyzer <sup>3</sup>http://www.basic.northwestern.edu/biotools/oligocalc.html yeasts using the lithium acetate method (Gietz and Schiestl, 2007). The yeast strains used in this study were DY1457 (MATa, ade1 can1 his3 leu2 trp1 ura3), the mutant ZHY3 - 1zrt1/zrt2 (DY1457 + zrt1::LEU2, zrt2::HIS3) defective in high and low affinity zinc uptake, and 1fet3fet4 (MATa trp1 ura3 Dfet3::LEU2 Dfet4::HIS3), defective in high and low affinity iron uptake system. Yeast strains were grown on liquid synthetic complete medium (SC-URA-MET/Glu) of the following composition: yeast nitrogen base supplemented with amino acids (without uracil and methionine), 2% (w/v) glucose, pH 5.3 (containing 0.2 mM Zn) overnight at 30◦C with shaking. On the next day the OD<sup>600</sup> was measured, adjusted to OD<sup>600</sup> of approximately 0.2 and yeasts were grown for another 2–5 h. The OD<sup>600</sup> was measured again, adjusted to OD = 0.3, series of dilutions were made (1.0, 0.1, 0.01, 0.001, and 0.0001) and 3 µl aliquots of each yeast culture were spotted onto plates containing (SC-URA-MET/Glu) medium solidified with 2% (w/v) agar supplemented with components depending on needs. To determine whether Zn is a substrate for the NtZIP1 like, the 1zrt1/zrt2 yeast strain with the expression of pUG35, pUG35-NtZIP1-like, pUG35-NtZIP1-like-EGFP, pUG36 or pUG36-EGFP-NtZIP1-like and WT (DY1457) with the expression of pUG35 or pUG36 (empty vector) were grown on liquid SC-URA-MET/Glu medium (containing 0.2 mM Zn) and spotted onto the agar-solidified SC-URA-MET/Glu medium containing series of EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N<sup>0</sup> ,N0 -tetraacetic acid) concentrations: 2.5, 5.0. 7.5, and 10.0 mM. Yeast growth was monitored for the next 5 days. To examine if Cd is a substrate for the NtZIP1-like, yeast WT (DY1457) was transformed with the pUG35, pUG35-ZIP1-like and pUG35-ZIP1-like-EGFP, and spotted onto the agar-solidified SC-URA -MET/Glu medium containing range of Cd (as CdCl2) concentrations (5, 10, 20, 50, and 75 µM). The sensitivity to cadmium was monitored. To determine whether Fe is a substrate for the NtZIP1 like, complementation of the growth defect of 1fet3fet4 mutant line by expression of NtZIP1-like (the same constructs as above were used for expression) was tested on plates containing agar-solidified SC-URA-MET/Glu control medium. Moreover, modification of the sensitivity to high Fe due to expression of NtZIP1-like was examined on medium supplemented with 50 or 100 µM FeCl3. ### Subcellular Localization of NtZIP1-Like Protein The entire cDNA sequence of the ORF of NtZIP1-like were obtained using Phusion polymerase and primers introducing CACC at the 5<sup>0</sup> end of the amplicon (underlined): forward 5 <sup>0</sup> CACCATGAATAACCACAATGTCCAAGT 3<sup>0</sup> and reverse 5 0 -AGCCCATTTAGCCATCACAGA -3<sup>0</sup> . The CACC overhand in the forward primer is required for directional cloning in the pENTR/D TOPO <sup>R</sup> vector (add provider). Following amplification, the cDNA was ligated into a Gateway entry vector pENTR/D-TOPO (Invitrogen). Fusion proteins with GFP were produced by the recombination (LR reaction) of entry vectors pENTR/D-TOPO-NtZIP1-like with destination vector pMDC43 (N-terminal GFP) (Curtis and Grossniklaus, 2003) using the Gateway system (Invitrogen). Resulting construct pMDC43-GFP-ZIP1-like was sequenced (Genomed, Poland), then used for determination of the subcellular localization of the NtZIP1-like in tobacco cells. The pMDC43-GFP-ZIP1-like fusion protein was transiently expressed in tobacco leaves as described by Siemianowski et al. (2013). Leaves of 6-week-old WT tobacco grown on control medium were infiltrated with Agrobacterium tumefaciens carrying the pMDC43-GFP-ZIP1-like construct. Three days from the infiltration, leaves were analyzed using a Nikon A1 confocal laser scanning microscope (Melville, NY, United States). GFP signals were detected by excitation with the 488 nm line of the argon laser and emission was recorded between 500 and 560 nm. To confirm plasma membrane localization of NtZIP1-like, cell walls at the plasma membrane border of examined tobacco epidermal cells were visualized by staining with the 50 µM water solution propidum iodide (20 min), a membrane-impermeant red fluorescent dye (Suh et al., 2007; McFarlane et al., 2010). Imaging was detected by 543 nm excitation and 617 nm emission. In parallel, chlorophyll autofluorescence was monitored using a HeNe (543 nm) laser for excitation. ### Hydroponic Experiments Developmental Regulation of NtZIP1-Like Expression To study the organ-specific expression of NtZIP1-like which depends on a developmental stage of the vegetative phase of growth the 3-week old tobacco plants were transferred from the agar plates to the control liquid medium (see section "Plant Material and Growth Conditions") and cultivated for up to 6 weeks. For the first 3 weeks on hydroponics the nutrient solution was changed every 3–4 days (plants were grown in 2- L plastic pots, five plants per pot). Next they were transferred to 1.2-L pots (two plants per pot) for the consecutive 3 weeks, and the medium was changed every 2nd day. The plant samples were collected at three stages of vegetative development: (Stage 1) small seedlings (3 weeks at the plates and 1 week on hydroponics); (Stage 2) young plants with rosette leaves (3 weeks on plates and 3 weeks on hydroponics); (Stage 3) adult plants with formed stem (3 weeks on plates and 6 weeks on hydroponics). At the Stage 1 and Stage 2 all leaf blades and all roots were collected separately. At the Stage 3 the following organs were collected: (i) from the aerial part of each plant – (a) two young leaves counting from the top (length of the blade of the smallest one was 0.5 cm); (b) two oldest leaves (counting from the base); (c) stem −3 cm of the middle part; (ii) rootstwo segments of the roots which grew out directly from the hypocotyl (adventitious roots were not included into analysis): (a) apical segment: 3–4 cm measured from the tip of the root; (b) basal segment: 3–4 cm measured from the base of the root. Plant samples were immediately frozen in the liquid nitrogen and stored in −80◦C until expression analysis. Three independent biological replicate experiments were performed. For each repetition samples were collected from a total of 30 plants (for Stage 1), 15 plants (for Stage 2) and 10 plants (for Stage 3). ### Regulation of the Expression of NtZIP1-Like by Zinc To determine if the expression of NtZIP1-like depends on Zn availability, the 5-week old plants (see section "Plant material and growth conditions") were grown for the next 4 days in the control medium, then they were subjected to the following treatments: (i) to Zn deficit (Zn was omitted from the medium) for 4 days; (ii) to Zn deficit for 4 days followed by re-supply with control conditions for 2 days; (iii) to Zn excess (50 µM Zn present in the control medium) for 1 day; (iv) control medium in parallel to all treatments. At the end of each experiment plant material was collected, frozen in liquid nitrogen, and stored in −80◦C for expression analysis. The following organs were collected: (i) the blades of the 2nd and 3rd leaf (counting from the base) without petioles and the major midribs; (ii) two sectors of the roots which grew out directly from the hypocotyl (adventitious roots were not included into analysis): (a) 3–4 cm of the apical region; (b) 3–4 cm of the basal region. Three independent biological replicate experiments were performed. Samples were collected from a total of 10 plants for each repetition. ### Statistical Analysis All presented data are from one experiment that is representative of three to four independent replicate experiments. Statistical significance was evaluated at the 0.05 probability level using Student's t-test. ### RESULTS ### Bioinformatic Analysis of Transporter Families in Tobacco Arabidopsis thaliana cDNAs from major metal transport families were used as the query sequences to identify genes encoding Zn transporters in tobacco. By screening the tobacco genome scaffolds, sequences of tobacco genes that significantly matched with the query cDNAs (query coverage > 80%) were selected. The following protein families from A. thaliana were included in the search: (i) ZIPs: ZRT, IRT-like proteins; (ii) NRAMPs: natural resistance-associated macrophage proteins; (iii) MTPs: metal tolerance proteins; (iv) MRP/ABCC: multidrug resistance proteins. For each gene from A. thaliana used as a query, several tobacco homologous sequences were identified on different scaffolds. These sequences were screened for exon orientation, start and end positions, and confidence scores for the boundaries (Supplementary Table S2). Further the selected tobacco scaffolds were screened to identify full putative genomic sequences of NtZIP, NtNRAMP, NtMTP, and NtMRP (ABCC) genes, including transcription start sites, exons, introns, and polyadenylation sites using the FGENESH tool (Salamov and Solovyev, 2000), whereas Phobius system based on a hidden Markov model (HMM) approach, was applied to predict membrane topology of NtZIP1-like protein. The list of genomic sequences comprises twenty-one newly identified tobacco putative metal transporters. Their names were given according to the NCBI terminology of the genes from A. thaliana, which were used as a query (Supplementary Figure S2). Identified sequences (Supplementary Figure S2) were used to design primer pairs (Supplementary Table S1) for determination of their transcript level in the leaf blades of tobacco plants exposed to high Zn. ### Response of Genes from the ZIP, NRAMP, MTP, and MRP/ABCC Families in Tobacco Leaves to High Zn To determine which of the identified metal transporters could be potentially involved in the regulation of Zn in tobacco leaves, their expression in the blades of plants grown in the presence of 200 µM Zn for up to 3 days was compared to the control conditions (Figure 1). From the ZIP family, three genes were identified with a several-fold difference in the transcript level between the Zn-exposed plants relative to those grown at the control medium. The most significant change was noted for NtZIP1-like and NtZIP4 (downregulation) and NtZIP11 (upregulation). Within the NtNRAMPs elevated expression was detected for a putative transporter NtNRAMP3-like. Moreover, modified expression was noted for three isoforms of NtMTP2. Out of identified six putative MRP/ABCC transporters which were subjected to analysis, the expression of three of them (NtMRP10-like and at a lower level NtMRP5-like and NtMRP14 like) were modified by high Zn. ### Phylogenetic Relationship of NtZIP1-Like from Tobacco In this study, the focus was on finding genes potentially involved in the accumulation of Zn in tobacco leaves, which respond to high Zn. The ZIP family proteins are considered as major Zn uptake transporters (Ricachenevsky et al., 2015). Based on downregulation of NtZIP1-like by high Zn in leaves (Figure 1), the assumption was made that it plays a role in Zn influx into the cytosol. Therefore, the NtZIP1-like was chosen for cloning and characterization. The ORF of the new tobacco ZIP family member – NtZIP1 like, consists of 1104 bp (Table 1) with 3 exons (Figure 2), and according to the prediction made by the program Fgenesh encodes 367 amino acids. To define the evolutionary relationship between ZIP1-like and the ZIP1 proteins from other organisms, as well as the other ZIPs, a phylogenetic tree was constructed (Figure 3). It included ZIP proteins from three species of tobacco (NtmZIP1-like, NsZIP1-like and NaZIP1-like), from A. thaliana, M. truncatula, and V. vinifera. It shows that NtZIP1-like is most closely related to ZIP1 proteins from other organisms including tobacco (NtmZIP1-like, NsZIP1-like and NaZIP1-like), Medicago truncatula (MtZIP1), Vitis vinifera (VvZIP1), and A. thaliana (AtZIP1). Within all ZIP1 sequences under comparison, NtZIP1 (Sano et al., 2012) formed a distinct clade with MtZIP3 and MtZIP4 from M. truncatula. The alignment of protein sequences defined at the phylogenetic tree as the closest homologs showed that the structure of NtZIP1-like is in agreement with the structure of other ZIP family members (Grotz and Guerinot, 2006). It contains eight transmembrane domains (TMs), a longer N-terminal region, a very short C tail, and a cytosolic variable region between TM domains III and IV (Figure 4). Histidine residues in the TMs II, IV, and V are highly conserved throughout the entire ZIP family. Our sequence analysis shows Expression under control conditions was set to 1 as the frame of reference within each experiment. Values correspond to means ± SD (n = 3); those significantly different from the control (Student's t-test) are indicated by an asterisk (P ≤ 0.05). that NtZIP1-like exhibits high amino acid sequence similarity with AtZIP1 and other known ZIP family members within these three mentioned TM domains (Figure 4). Among them, amino acid sequence conservation within the signature region in the fourth TM domain was found. It contains consensus sequences (including a fully conserved histidine residue). On the other hand, a potential metal-binding motif containing multiple histidine residues present in the variable region between TM III and IV, differs between examined proteins primarily in the number of his residues and their localization. In this region, eight histidine residues were found in NtZIP1-like compared to nine present for example in AtZIP1, and only three in NtZIP1 (Figure 4). The NtZIP1 protein (Sano et al., 2012) formed also a separate clade containing AtZIP3 and MtZIP3, MtZIP4 and AtZIP5. The NtZIP1-like shares 56 and 54% identity at the amino acid level with AtZIP1 and NtZIP1, respectively, whereas 58 and 62% at the nucleotide level. The highest homology was found between the NtZIP1-like and other three tested tobacco ZIP1 proteins such as NtnZIP1-like (100%), NaZIP1-like (94 and 96%) and NsZIP1-like (95 and 97%, respectively (Table 1). ### NtZIP1-Like Localizes to the Plasma Membrane To gain insight into the functioning of NtZIP1-like, its subcellular localization was determined by transient expression of the NtZIP1-like protein fused to the N terminus of green fluorescent protein (GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter in tobacco leaves. FIGURE 3 \| Phylogenetic analysis of ZIP1 transporters from selected species. The unrooted tree was constructed based on amino acid sequences identified in the Aramemnon (Arabidopsis thaliana) and NCBI database (Nicotiana species, Medicago truncatula, Vitis vinifera), using the MEGA 7.0 software. The lengths of branches are proportional to the degree of divergence. Numbers in the figure represent bootstrap values (1000 replicates). The accession numbers are as follows: Arabidopsis thaliana, AtZIP1 - At3g12750.1, AtZIP2 - At5g59520.1, AtZIP3 - At2g32270.1, AtZIP4 - At1g10970.1, AtZIP5 - At1g05300.1, AtZIP6 - At2g30080.1, AtZIP7 - At2g04032.1, AtZIP8 - At5g45105.1, AtZIP9 - At4g33020.1, AtZIP10 - At1g31260.1, AtZIP11 - At1g55910.1, AtZIP12 - At5g62160.1; Nicotiana attenuata, NaZIP1-like XP\_019256554; Nicotiana tabacum, NtZIP1-like XP\_016507999, NtZIP1- NP\_001312674; Nicotiana sylvestris: NsZIP1-like XP\_009772024; Nicotiana tomentosiformis, NtomZIP1-like XP\_009608181; Medicago truncatula: MtZIP1 - AAR08412.1, MtZIP2 - AAG09635, MtZIP3 - AY339055, MtZIP4 - AY339056, MtZIP5 - AY339057, MtZIP6 - AY339058, MtZIP7 - AY339059; Vitis vinifera, VvZIP1 - XP\_002264603.2. Three days after the infiltration of the leaves with Agrobacterium expressing pMDC43-GFP-ZIP1-like, the GFP signal (green fluorescence) was detected in tobacco epidermal cells along the cell walls indicating localization of NtZIP1-like protein at the plasma membrane (Figure 5). Cell walls were stained with propidium iodide and the green fluorescence (Figure 5A) coincided with the red signal, derived from propidium iodide (Figures 5B,C). The co-localization of the GFP-derived signal and propidium iodide staining of the cell walls indicates localization of GFP-fused NtZIP1-like protein at the plasma membrane (Pighin et al., 2004; Lee et al., 2010; Siemianowski et al., 2013). Moreover, at higher magnification the signal from the cell wall (red) and from the GFP-labeled plasma membrane (green) were separated (indicated by arrows; Figures 5E–G). Altogether, results support the conclusion that NtZIP1-like is a plasma membrane protein. ### Yeast Complementation Supports a Role for NtZIP1-Like as a Zn Transporter Yeast functional complementation was used to determine the capacity of NtZIP1-like to transport Zn. The yeast zrt1zrt2 double mutant (ZHY3) defective in high and low affinity Zn uptake was used (Eide, 2003). The expression of fulllength cDNA of NtZIP1-like gene fused with the eGFP at its C-terminal end (construct pUG35EGFP-NtZIP1-like-EGFP), as well as at its N-terminal end (construct pUG36-EGFP-NtZIP1 like) did not complement the growth defect of the 1zrt1zrt2 yeast mutant (Figure 6A). In contrast, the expression of the construct pUG35-NtZIP1-like (with the STOP codon) fully restored growth under Zn-limited conditions (Figure 6A). These result indicates that NtZIP1-like is a plasma membrane protein mediating Zn uptake. The lack of rescue by constructs containing EGFP both at the C- and N-terminal end suggests that the presence of the eGFP protein makes the NtZIP1-like protein dysfunctional. Some ZIP proteins mediate transport of Cd (Ramesh et al., 2003; Nakanishi et al., 2006; Stephens et al., 2011). To determine if NtZIP1-like is a Cd uptake protein, the wild-type yeast line DY1457 was transformed with pUG35-NtZIP1-like (with the STOP codon), and pUG35-NtZIP1-like-EGFP. If the NtZIP1-like is involved in Cd influx, the yeast transformants should be more sensitive to this metal. As shown in Figure 6B the growth of the wild-type transformed with the empty vector or with both tested constructs was limited by a range of Cd present in the medium to the same extent indicating no Cd transport capacity by NtZIP1-like. Finally, to study the capacity of the NtZIP1-like to transport Fe, a yeast mutant 1fet3fet4 defective in both high- and low affinity Fe uptake systems was transformed with the NtZIP1-like cDNA to examine if it complements the defect in Fe transport. As shown in Figure 6C, expression of NtZIP1-like in the 1fet3fet4 did not restore the growth of mutants at control conditions, and it did not modify the sensitivity of yeast to Fe excess. To conclude, the results indicate that the NtZIP1-like does not transport Fe. ### Developmental Regulation of NtZIP1-Like Our study showed that NtZIP1-like is expressed both in the roots, leaves and stems, but the level depends on the developmental stage (Figure 7). The transcript level in the leaves was very low in young 4-week old plants compared to a 6-fold increase in 6 week old tobacco. In the adult 9-week old plants its expression in young leaves was 3-times higher than in the old ones. NtZIP1-like was expressed at a moderate level in stems. In the roots of young 4-week old plants the transcript level was very low, and a 6-fold increase was detected in 6-week old tobacco. It remained at that level in adult 9-week old plants and did not differ in the apical and basal part of the root. ## Expression of NtZIP1-Like Is Zn Regulated It has been shown that Zn is a substrate for NtZIP1-like. To know more about the possible physiological role of NtZIP1-like in tobacco, its expression was analyzed in the roots, leaves and stems of plants exposed to Zn excess (50 µM for 1 day), and to Zn-deficiency (no Zn for 4 days) subsequently followed by a replete conditions (4-day Zn deficit followed by 2 days of control conditions). In agreement with downregulation in the leaf blades by 200 µM Zn (Figure 1), downregulation by 1-day exposure to 50 µM Zn was detected in leaves, and in the roots (both in the apical and basal segment) (Figure 8). Interestingly, its expression was highly upregulated by Zn-deficiency in the leaves and in the basal segment of the roots. No response to low Zn in the medium was noted in the apical part of the root considered as primarily responsible for Zn uptake (Figure 8). signal from the cell wall (stained red) clearly visible between adjacent GFP-labeled plasma membranes (arrows). ### DISCUSSION Although tobacco, as a plant with a high capacity to accumulate large amount of metals (including Zn and Cd) in leaves, is used for phytoremediation of metal contaminated soil (Herzig et al., 2003, 2014; Lugon-Moulin et al., 2004; Dguimi et al., 2009; Vangronsveld et al., 2009), metal transporters involved in uptake and storage of metals in leaf tissues remain unknown. Here, based on bioinformatics searches for tobacco metal transporter sequences (Supplementary Figure S2 and Supplementary Table S2), and subsequent analysis of the regulation of the candidate genes by Zn excess (200 µM Zn) in leaves, ten genes (out of twenty-one tested) with significantly modified expression were identified (Figure 1). They represent putative metal transport genes that likely contribute to the storage of Zn excess in tobacco leaves, and include transporters involved in sequestration, redistribution and uptake of metals. In sequestration of Zn in tobacco leaves exposed to 200 µM Zn three isoforms of NtMTP2 may play a role. Elevated expression of two isoforms of NtMTP2-X1 and NtMTP2-X2 (Figure 1C) suggests a likely involvement in loading of Zn into vacuoles, which are the major storage compartments within cells. The MTP2 proteins are not fully characterized so far in plants. It is known that the MTP2 belongs to the Group 1 of MTP vacuolar Zn transporters. Phylogenetic analysis showed that MTP1, MTP2, and MTP3 originate from a common MTP1/2/3 ancestors (Gustin et al., 2011). The concentration of a metal in vacuoles depends not only on efficient loading, but also on the rate of mobilizing vacuolar pool back to the cytosol, which, among others, is under control of NRAMP proteins. In tobacco leaves expression of NtNRAMP3 like was significantly induced by high Zn supply (Figure 1B). Oomen et al. (2009) showed that TcNRAMP3/4 (and to a lesser extent AtNRAMP3/4) expression is regulated by Zn supply (lowefficient-excess), though the pattern of the regulation has not been fully established. However, until now Zn has not been shown to be a substrate for NRAMP3. The AtNRAMP3 from A. thaliana and TcNRAMP3 from T. caerulescens mediate efflux of Fe, Cd, and Mn from vacuoles to the cytoplasm (Thomine et al., 2003; Oomen et al., 2009). Ability to transport not only Fe, Mn, Cd but also Zn was shown for AtNRAMP4 and TcNRAMP4 only (Oomen et al., 2009). Thus, future studies will show whether the NtNRAMP3-like is localized to the tonoplast (like AtNRAMP3 or TcNRAMP3) or to the plasma membrane (like e.g., OsNRAMP3; Yamaji et al., 2013), what the substrates are, and what its role in the accumulation of high amounts of Zn in tobacco leaves. The next identified new putative metal transporters regulated upon high Zn concentration in tobacco leaves are from the MRP/ABCC family. The major changes were found for the NtMRP10-like and NtMRP14-like, whereas to a lesser extent for NtMRP2-like, NtMRP3-like and NtMRP5-like (Figure 1D). The MRP/ABCC proteins carry various xenobiotics including metal complexes. Until now, there are only a few studies on plants indicating involvement of MRP/ABCC proteins in the transport of metals as conjugates to various substrates (Klein et al., 2006). Heterologous expression of AtMRP7 in tobacco has suggested a role in Cd transport into the root vacuoles (Wojas et al., 2009). AtABCC1 and AtABCC2 were shown to be targeted to the tonoplast and mediated the vacuolar sequestration of phytochelatin (PC) complexes with Cd(II) and Hg(II) (Park et al., 2012). The MRP/ABCC genes have been shown to be regulated by metals. For example, the expression of AtMRP3 is induced by Cd, Ni, As, Co, and Pb, but not Zn or Fe (Bovet et al., 2003; Zientara et al., 2009). Upregulation by Cd was also confirmed for AtMRP6 (Gaillard et al., 2008) and AtMRP7 (Bovet et al., 2003), and by high Zn for TcMRP10 in the roots and shoots of Zn hyperaccumulator T. caerulescens (Hassinen et al., 2007). Identification in tobacco leaves of such Zn-responsive MRP/ABCC genes is important for future study on the regulation of Zn homeostasis upon treatment with high Zn. The emphasis in this study was to shed more light on the regulation of Zn acquisition by cells in the leaves. The ZIP proteins constitute a major Zn uptake system (Sinclair and Krämer, 2012). Here, the NtZIP1-like was cloned and characterized to better understand its function in tobacco. NtZIP1-like contains an ORF of 1104 bp, encoding a predicted protein of 367 amino acids (Table 1). Phylogenetic analysis of the ZIP family proteins shows that the NtZIP1-like forms a distinct clade with other ZIP1 proteins from three tobacco species (NatmZIP1-like, NsZIP1-like, and NaZIP1-like), A. thaliana, M. truncatula and V. vinifera (Figure 3). Sequence comparisons (Figure 4) showed that the deduced NtZIP1-like protein shares three independent experiments. FIGURE 8 \| Expression pattern of NtZIP1-like in N. tabacum under various Zn conditions. Plants were grown in standard nutrient solution (control) and then transferred into modified control media: supplemented with 50 µM Zn for 1 day (1d); without Zn for 4 days (4d - Zn deficiency); plants grown at Zn-deficiency conditions for 4 days were transferred to the control medium for 2 days (6d - Zn replete). RT-qPCR analyses was performed on cDNA prepared from leaves (L), apical part of roots (AR) and basal part of roots (BR) of N. tabacum. Gene expression was normalized to the PP2A level. Values correspond to means ± SD (n = 3); those significantly different are indicated by an asterisk (P ≤ 0.05). all the basic characteristic features of members of the ZIP family of metal transporters. It has eight TM domains, a long N-terminal end, a very short C tail, and a cytoplasmic variable region between TM III and IV (Figure 4). The variable region contains a histidine rich domain (HRD) with the motif (HX)<sup>n</sup> (n = 2, 3, and 4) which has been proposed as a metal binding site. The characteristic feature of ZIP proteins is the presence of a signature motif within the TM IV, and highly conserved histidine residues in TM domains II, IV, and V (Eide et al., 1996; Eng et al., 1998; Grotz et al., 1998; Guerinot, 2000; Rogers et al., 2000; Gaither and Eide, 2001). They all are present in the NtZIP1-like (Figure 4). Analysis showed that two tobacco ZIP1 proteins – newly cloned NtZIP1-like and NtZIP1 (Sano et al., 2012), do not cluster together (Figure 3). They share 54% identity at the amino acid level (Table 1). Comparison of NtZIP1-like and NtZIP1 amino acids sequences (Figure 4) showed that an important difference between them lies within a variable cytoplasmic HRD region between the TM III and IV. Although this region displays low sequence conservation among ZIP metal transporters, most of them contain the motif (HX)<sup>n</sup> (n = 2, 3, and 4), which has been proposed as a metal binding site (Eide et al., 1996; Eng et al., 1998; Grotz et al., 1998). Only three (HX) repetitions were detected in NtZIP1, whereas eight in the newly cloned NtZIP1-like. To compare, other ZIP1 proteins contain eight or nine (HX) repetitions. The exact function of the loop between TM III-IV is yet to be determined, however, a study by Nishida et al. (2008) on the TjZNT1 ZIP transporter from Thlaspi japonica showed that deletion of a part of the HRD region containing his residues localized closer to the TM IV (HRD, position 207–217 aa) abolished Zn transport ability. Hence a difference in the structure at the amino acid level might contribute to a different substrate specificity. NtZIP1 and NtZIP1-like do seem to have different substrate specificities. The expression of NtZIP1 in yeast significantly enhanced Fe accumulation suggesting Fe uptake activity (Sano et al., 2012). In contrast, the NtZIP1-like failed to alter the Fe-limited growth defect of fet3fet4 yeast mutant indicating it may not transport Fe (Figure 6C). NtZIP1-like is also unlikely to mediate Cd uptake since its expression in WT yeast did not modify the sensitivity to Cd (Figure 6B). Functional complementation of the zrt1zrt2 mutant, defective in Zn uptake supports its potential role as a Zn transporter (Figure 6A). NtZIP1-like localizes to the plasma membrane when transiently expressed in tobacco (Figure 5). Therefore, our results indicate that NtZIP1-like is a tobacco ZIP1 uptake protein for Zn, but not for Cd or Fe. In general, an ability of ZIP1 proteins to transport Fe was shown for PtZIP1 (Fu et al., 2017). More Fe transporters were identified among other ZIP proteins for example ZmZIP2-8, OsZIP5 and OsZIP8 (Li et al., 2013), MtZIP3, 5, 6 (López-Millán et al., 2004), PtZIP7 (Fu et al., 2017) and NtZIP1 (Sano et al., 2012). Expression of the NtZIP1-like was detected in all plant organs, which suggests rather a universal role in maintaining Zn homeostasis (Figure 7). Its role seems to be more pronounced at later developmental stages. The transcript level is greater in older plants, especially in younger leaves (as compared with the older ones) suggesting a contribution to supplying cells in developing organs with Zn. Analysis of the regulation of the NtZIP1-like expression by Zn availability showed upregulation by Zn-deficiency in the roots and leaves (Figure 8), which was similar to AtZIP1 and OsZIP1 (Ramesh et al., 2003; Milner et al., 2013). Interestingly, in the roots upregulation of NtZIP1-like was limited to the basal segment of the root only, and was not detected in the apical part. It is known that the young, apical segment of the root is responsible for acquisition of nutrients, however, not much is known about the role of the older, basal region. Our studies clearly indicate that NtZIP1-like is a Zn-deficiency inducible Zn uptake transporter in leaves and in roots (though in roots the induction takes place only in the basal part; Figure 8). Further research is needed to demonstrate the NtZIP1-like tissue-specific expression and regulation, as it is not clear if it is involved in Zn acquisition from the medium, or in internal uptake. Distinct regulation of ZIP genes in a different root sectors has been shown also in rice (Ishimaru et al., 2005). Expression of OsZIP4 was detected in the meristematic region of the Zn-deficient roots. Similarly, AtZIP2p::GUS expression analysis revealed higher induction in the younger region of the roots grown under nutrient-replete conditions, as compared to a lower induction nearer the mature part at the root-shoot junction (Weber et al., 2004). In general, it is known that upregulation upon Zn deficiency conditions and downregulation in replete medium is ascribed to genes involved in the acquisition of micronutrients (Sinclair and Krämer, 2012), and these two features are characteristic for NtZIP1-like (Figure 8). It is known that in the leaves of tobacco plants exposed to Zn excess, the metal is not distributed equally throughout the mesophyll cells. Instead, high concentrations were found in clusters of adjacent cells (Zn-accumulating cells) in contrast to its low level in neighboring non-accumulating ones (Siemianowski et al., 2013). Distinct expression patterns of Zn transport genes must underlie such different Zn uptake and accumulation capacity. Knowing this, we searched for genes differentially regulated in the leaves by high Zn. The NtZIP1-like was identified initially as downregulated by 200 µM Zn (Figure 1), and confirmed later as downregulated by 50 µM Zn (Figure 8). We hypothesize that the downregulation observed in leaves upon Zn excess could be a part of the molecular mechanism occurring in the low Zn-accumulating cells that prevents them from excessive uptake of Zn. Further comparative studies on the regulation of NtZIP1-like expression in leaves at low and high Zn at the cellular and tissue level are necessary in the future to investigate this. ### CONCLUSION The bioinformatics analysis using information from the tobacco genome and the detailed expression study has led to the identification of ten new tobacco putative transporters involved in the regulation of Zn accumulation in tobacco leaves. They belong to different major families of metal transporters (ZIP, NRAMP, MTP, and MRP/ABCC), and undergo differential regulation in the leaves of tobacco plants exposed to 200 µM Zn. The upregulation of NtZIP11-like, NtNRAMP3, three isoforms of NtMTP2, three MRP/ABCC genes such as NtMRP10-like, NtMRP5-like and NtMRP14 like, and downregulation of NtZIP1-like and NtZIP4, indicate their contribution to a range of processes underlying uptake, sequestration and redistribution of metals in the cells and tissues. These data provide an important input for further research on metal homeostasis mechanisms in tobacco, the species used for phytoremediation of metal contaminated soil. The detailed study on the newly cloned NtZIP1-like showed that the encoded protein is localized to the plasma membrane and mediates uptake of Zn, but not Fe or Cd. It is expressed in the roots and leaves – but the level of the transcript depends on the developmental stage. It is also regulated by the availability of Zn, being highly up-regulated by Zn-deficiency specifically in the leaves and in the basal part of the root but not in the apical zone. We have shown previously that tobacco mesophyll cells have a distinct capacity to store Zn in the "Zn-accumulating cells" which are next to non-accumulating ones in the leaf blade (Siemianowski et al., 2013). Our detailed studies on the NtZIP1-like suggest that it might be a candidate gene involved in the restriction of Zn uptake by the mesophyll cells with low capacity to accumulate Zn. ### AUTHOR CONTRIBUTIONS AP carried out all experiments. KK was involved in yeast study, cloning and expression analysis. MK was involved in cloning, expression analysis and hydroponic experiments. AB contributed to expression analysis. MP was involved in bioinformatics analysis and hydroponic experiments. JT performed bioinformatics analysis. BP contributed to confocal analysis. LW supervised yeast complementation assays. DA designed the study concept, coordinated the research and supervised experiments, performed data analysis, and wrote the manuscript. All authors read and approved the final manuscript. ### FUNDING This work was financially supported by National Science Center, Poland (Grant HARMONIA No. NZ3/00527). ### REFERENCES ### ACKNOWLEDGMENTS We would like to thank Dr. Rafał Milanowski (Department of Molecular Phylogenetics and Evolution, Faculty of Biology, University of Warsaw Biological and Chemical Research Centre) for advice and comments on the construction of the phylogenetic tree. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00185/ full#supplementary-material legume Medicago truncatula. Biometals 24, 51–58. doi: 10.1007/s10534-010- 9373-6 in rice. Nat. Commun. 4, 2442–2453. doi: 10.1038/ncomms 3442 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Papierniak, Kozak, Kendziorek, Barabasz, Palusinska, Tiuryn, ´ Paterczyk, Williams and Antosiewicz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Biofortified Crops Generated by Breeding, Agronomy, and Transgenic Approaches Are improving Lives of Millions of People around the world Monika Garg\, Natasha Sharma, Saloni Sharma, Payal Kapoor, Aman Kumar, Venkatesh Chunduri and Priya Arora* National Agri-Food Biotechnology Institute, Mohali, Punjab, India #### Edited by: Felipe Klein Ricachenevsky, Universidade Federal de Santa Maria, Brazil #### Reviewed by: Hannetz Roschzttardtz, Pontificia Universidad Católica de Chile, Chile Michael La Frano, California Polytechnic State University, United States Ümit Barıs¸ Kutman, Gebze Technical University, Turkey #### \Correspondence:* Monika Garg [email protected], [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Nutrition Received: 09 August 2017 Accepted: 29 January 2018 Published: 14 February 2018 #### Citation: Garg M, Sharma N, Sharma S, Kapoor P, Kumar A, Chunduri V and Arora P (2018) Biofortified Crops Generated by Breeding, Agronomy, and Transgenic Approaches Are Improving Lives of Millions of People around the World. Front. Nutr. 5:12. doi: 10.3389/fnut.2018.00012 Biofortification is an upcoming, promising, cost-effective, and sustainable technique of delivering micronutrients to a population that has limited access to diverse diets and other micronutrient interventions. Unfortunately, major food crops are poor sources of micronutrients required for normal human growth. The manuscript deals in all aspects of crop biofortification which includes—breeding, agronomy, and genetic modification. It tries to summarize all the biofortification research that has been conducted on different crops. Success stories of biofortification include lysine and tryptophan rich quality protein maize (World food prize 2000), Vitamin A rich orange sweet potato (World food prize 2016); generated by crop breeding, oleic acid, and stearidonic acid soybean enrichment; through genetic transformation and selenium, iodine, and zinc supplementation. The biofortified food crops, especially cereals, legumes, vegetables, and fruits, are providing sufficient levels of micronutrients to targeted populations. Although a greater emphasis is being laid on transgenic research, the success rate and acceptability of breeding is much higher. Besides the challenges biofortified crops hold a bright future to address the malnutrition challenge. Keywords: malnutrition, biofortification, transgenic, agronomic, breeding ### INTRODUCTION "Biofortification" or "biological fortification" refers to nutritionally enhanced food crops with increased bioavailability to the human population that are developed and grown using modern biotechnology techniques, conventional plant breeding, and agronomic practices. The United Nations Food and Agriculture Organization has estimated that around 792.5 million people across the world are malnourished, out of which 780 million people live in developing countries (1). Apart from this, around two billion people across the world suffer from another type of hunger known as "hidden hunger," which is caused by an inadequate intake of essential micronutrients in the daily diet (2, 3) despite increased food crop production (4). Besides this overnutrition is growing matter of concern. So far, our agricultural system has not been designed to promote human health; instead, it only focuses on increasing grain yield and crop productivity. This approach has resulted in a rapid rise in micronutrient deficiency in food grains, thereby increasing micronutrient malnutrition among consumers. Now agriculture is undergoing a shift from producing more quantity of food crops to producing nutrient-rich food crops in sufficient quantities. This will help in fighting "hidden hunger" or "micronutrient malnutrition" especially in poor and developing countries, where diets are dominated by micronutrient-poor staple food crops (5). 282 Traditionally, vitamins and minerals have been provided to the masses through nutrient supplementation programs, but it falls short of the goals set by the international health organizations as the supplementation programs rely on external funding that is not guaranteed to be available from year to year. Other limitations are purchasing power of poor people, their access to markets and health-care systems, and lack of awareness regarding the longterm health benefits of these nutrient supplements (6, 7). Hence, biofortification of different crop varieties offers a sustainable and long-term solution in providing micronutrients-rich crops to people. Furthermore, biofortified crops with increased bioavailable concentrations of essential micronutrients are deployed to consumers through traditional practices used by agriculture and food trade which therefore provides a feasible way of reaching undernourished and low income group families with limited access to diverse diets, supplements, and fortified foods. From an economic viewpoint, biofortification is a one-time investment and offers a cost-effective, long-term, and sustainable approach in fighting hidden hunger because once the biofortified crops are developed; there are no costs of buying the fortificants and adding them to the food supply during processing (8–14). Furthermore, in the next few decades, a major population increase might take place in the developing world and with the changing climatic conditions; achieving food security will pose a greater challenge (15, 16). Thus, organizations such as the World Health Organization and the Consultative Group on International Agricultural Research (CGIAR) have included the development of nutritionally enhanced high-yielding biofortified crops as one of their main goals (17). ### NECESSITY AND SOCIOECONOMIC DEVELOPMENT DERIVE BIOFORTIFICATION RESEARCH Humans require around 40 known nutrients in adequate amounts to live healthy and productive lives (Table 1). The mineral elements—sodium, potassium, calcium, magnesium, phosphorous, chlorine, and sulfur—are classified as essential nutrients that Table 1 \| Essential micro- and macronutrients required for good human health. are required in small amounts in the body. The other class of essential nutrients required in very small amounts in the human body are termed as micronutrients—namely iron, zinc, copper, manganese, iodine, selenium, molybdenum, cobalt, nickel, and vitamin A (18). Collectively, these nutrients play crucial roles in humans and dictate our physical and mental development (19). Many micronutrients act as cofactors for the functioning of various enzymes in the human body and thereby regulate important functions and metabolic processes in our body (20). For humans, agricultural products are the primary source of nutrients, especially for those living in developing countries (21–23). However, the diet of the population based on cereals such as rice, wheat, cassava, and maize contain insufficient amounts of several nutrients such as vitamin A, iron, zinc, calcium, manganese, copper, iodine, or selenium with respect to meeting daily requirements. These nutrient deficient agricultural products cannot support healthy lives and can result in poor health, sickness, increased morbidity and disability, impaired development, stunted mental and physical growth, diminished livelihoods, and reduced national socioeconomic development (24–29). Childhood stunting prevalent in many developing countries is associated with micronutrient malnutrition in children starting from fetal development to 4 years of age (25). Micronutrient deficiencies affect about 38% of pregnant women and 43% of pre-school children worldwide. More than 30% of the world's population has been reported to be anemic (30) and suffering from hidden hunger. The prevalence of anemia is more in developing countries compared with developed countries. Africa and South-East Asian countries are most affected (Figures 1A,B). Estimates have indicated that approximately half of this is attributed to iron deficiency (31). Hence, micronutrient malnutrition is the major challenge in many developing countries. Another important point of consideration is uneven distribution of the nutrients among different plant parts (32). For example, the iron content is high in rice leaves, but low in polished rice grain. Apart from under nutrition, growing incidence of overnutrition leading to problems of overweight and in particular, high rate of diabetes is a matter of concern. Consequently, biofortification is also directed toward enhancing the contents of desired micronutrients in the edible portion of crop plants. Nutritional targets for biofortification include elevated mineral content, improved vitamin content, increased essential amino acid levels, better fatty acid composition, and heightened antioxidant levels in crops (12). Biofortification of crop plants can provide enough calories to meet the energy needs along with providing all the essential nutrients needed for sound health. Furthermore, biofortifying the crops which are consumed by the poor population of the world can significantly improve the amount of nutrients consumed by this target population (33). ### BIOFORTIFICATION PATHWAY INCLUDES SEVERAL APPROACHES Producing nutritious and safe foods, sufficiently and sustainably, is the ultimate goal of biofortification (34). Biofortification of essential micronutrients into crop plants can be achieved through three main approaches, namely transgenic, conventional, and agronomic, involving the use of biotechnology, crop breeding, and fertilization strategies, respectively. Most of the crops targeted by transgenic, conventional breeding, and agronomical approaches include staple crops like rice, wheat, maize, sorghum, lupine, common bean, potato, sweet potato, and tomato (Figure 2). Cassava, cauliflower, and banana have been biofortified by both transgenic and breeding approaches while barley, soybean, lettuce, carrot, canola, and mustard have been biofortified with transgene and agronomic approaches. Higher numbers of crops have been targeted by transgenic means, while the practical utilization of biofortification is higher by breeding methods (Figures 3A,B). Cereals being staple crop have been targeted by all three approaches. Same is the case of legumes and vegetables. Interestingly, oil seed biofortification has been achieved through transgenic means, because limited availability of genetic diversity for the targeted component, low heritability, and linkage drag in the targeted crop (Figure 3B). Biofortification by breeding has been achieved in crops and specified components when genetic diversity is available in the utilizable form in the primary, secondary, or tertiary gene pool of the targeted crop. When genetic diversity is unavailable, genetic transformation is the better option. Transgenic-based approach has advantages that a useful gene once discovered, can be utilized for targeting multiple crops (Figure 4). Some important genes like phytoene synthase (PSY), carotene desaturase, nicotinamide synthase, and ferritin have been utilized in multiple events including multiple crops. In this manuscript, we have compiled the data from research to release on different food crops that are being targeted by the different approaches of biofortification. ### BIOFORTIFICATION THROUGH TRANSGENIC MEANS—MAXIMUM RESEARCHED AND MINIMUM UTILIZED Transgenic approach can be a valid alternative for the development of biofortified crops when there is a limited or no genetic variation in nutrient content among plant varieties (32, 35). It relies on the access to the unlimited genetic pool for the transfer and expression of desirable genes from one plant species to another which is independent of their evolutionary and taxonomic status. Furthermore, when a particular micronutrient does not naturally exist in crops, transgenic approaches remain the only feasible option to fortify these crop with the particular nutrient (7). The ability to identify and characterize gene function and then utilize these genes to engineer plant metabolism has been a key for the development of transgenic crops (36). Furthermore, pathways from bacteria and other organisms can also be introduced into crops to exploit alternative pathways for metabolic engineering (37). Transgenic approaches can also be used for the simultaneous incorporation of genes involved in the enhancement of micronutrient concentration, their bioavailability, and reduction in the concentration of antinutrients which limit the bioavailability of nutrients in plants. In addition, genetic modifications can be targeted to redistribute micronutrients between tissues, enhance the micronutrient concentration in the edible portions of commercial crops, increasing the efficiency of biochemical pathways in edible tissues, or even the reconstruction of selected pathways (38–40). Development of transgenically biofortified crops initially involves substantial amount of time, efforts, and investment during research and development stage, but in a long run, it is a cost-effective and sustainable approach, unlike nutrition-based organizational and agronomic biofortification programs (14, 19). Furthermore, genetic engineering has no taxonomic constraints and even synthetic genes can be constructed and used. Transgenic crops with enhanced micronutrient contents hold a potential to reduce micronutrient malnutrition among its consumers, especially poor people in developing countries (12). Numerous crops have been genetically modified to enhance their micronutrient Figure 3 \| Representation of reported biofortified crops by transgenic, agronomic, and breeding means. (A) Comparison of transgenic and breeding approaches of biofortification in terms of relative research and release of commercial crops. While higher emphasis is being laid on transgenic-based biofortification, success rate in terms of cultivar release is higher for breeding-based approach. (B) Percentage of different crops biofortified by different approaches. Cereals have been biofortified in largest number by all three biofortification approaches. Legumes and vegetables have also been targeted by all the approaches in almost equal percentage. Transgenic approach covers highest number of crops. Oilseed crops have been mainly targeted by transgenic approaches due to limited genetic variability. contents. Among micronutrients, vitamins, minerals, essential amino acids, and essential fatty acids have been targeted by the use of various genes from different sources to enhance the food crop nutritional level (Table 2). It has been found that PSY, carotene desaturase, and lycopene β-cyclase for vitamins, ferritin and nicotinamine synthase for minerals, albumin for essential amino acids, and Δ<sup>6</sup> desaturase for essential fatty acids have been widely reported as targets for biofortification (Figure 4). Successful examples of transgenic method are high lysine maize, high unsaturated fatty acid soybean, high provitamin A and iron carotene desaturase, nicotinamide synthase, and ferritin have been utilized in multiple events including multiple crops. rich cassava, and high provitamin A Golden rice. Reports are available for biofortified cereals, legumes, vegetables, oilseeds, fruits, and fodder crops. ### TRANSGENIC CEREALS ### Transgenic Rice (Oryza sativa) Rice has been targeted to address the global challenge of undernutrition. Vitamin deficiency is one of the major challenges #### Table 2 \| Tabulation of crops, nutrients, research status, and concerned publications on biofortification by transgenic means. (Continued) #### TABLE 2 \| Continued (Continued) #### TABLE 2 \| Continued Significant amount of information have been generated that hold a bright future to address the malnutrition challenge. that affect underprivileged population due to poor affordability. Golden Rice was an important breakthrough in this direction as an effective source of provitamin A (beta-carotene) with a significant potential to reduce disease burden by expressing genes encoding PSY and carotene desaturase (41–45). The level of beta-carotene precursor, i.e., phytoene, has been enhanced up to 23-fold by targeting gene encoding carotene desaturase (45). Folic acid (vitamin B9) is important for normal pregnancy and anemia (190). Rice has been genetically modified to increase folate content (up to 150-fold) by overexpressing genes encoding Arabidopsis GTP-cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase [ADCS (46, 47)]. The 100 g of modified rice was found to be sufficient to meet daily folate requirements of an adult individual. Rice has also been targeted to address the global challenge of iron deficiency anemia. Multiple reports have indicated an increase in iron content in rice by expressing genes encoding, nicotianamine aminotransferase (48), iron transporter OsIRT1 (49), nicotianamine synthase 1 (OsNAS1) and 2 (OsNAS2) (50–52, 191), soybean ferritin (52–54), and common bean ferritin (55). Iron biofortified rice was also synthesized by introducing multiple genes involved in iron nutrition (56–58). In addition to enhanced iron content, improvement in iron bioavailability was also achieved by reducing antinutrient compounds in rice such as phytic acid (59). Similarly, zinc content was also elevated in GM rice by overexpressing OsIRT1 (49) and mugineic acid synthesis genes from barley [HvNAS1, HvNAS1, HvNAAT-A, HvNAAT-B, IDS3 (60)]. Improvement in quality protein has been addressed by targeting essential amino acid content in rice by expressing seed-specific genes of bean β-phaseolin (61), pea legumin (62); Sesame 2S Albumin (63); soybean glycinin (64); bacterial aspartate kinase, dihydrodipicolinate synthase (DHPS) (65); maize DHPS (66); rice anthranilate synthase α-subunit (67); and E. coli aspartate aminotransferase (68). Rice has also been targeted for seed oil quality by increasing amount of polyunsaturated fatty acid that can help in the reduction of bad cholesterol levels in the body and improve human nutrition (192). An essential fatty acid α-linolenic acid has been enhanced in rice by expressing soybean omega-3 fatty acid desaturase (FAD3) gene [GmFAD3 (69)]. Flavonoids are associated with antioxidant activity and its content in rice has been enhanced by expressing maize C1 and R-S regulatory genes [Myb-type and basic helix-loop-helix-type transcription factors (70)]; and phenylalanine ammonia lyase and chalcone synthase (CHS) genes (71). To address the challenge of overnutrition and obesity, the content of less digestible and resistant amylose starch has been enhanced by expression of antisense waxy genes (72, 73) and antisense RNA inhibition of starch-branching enzymes (SBE) (74). Besides introducing micronutrients, expression of functional human milk protein (lactoferrin) in rice grains has opened the possibility for creating a value-added cereal-based ingredients that can be introduced into infant formula and baby food (75, 193). ### Transgenic Wheat (Triticum aestivum) Wheat is one of the most widely grown staple food crops in the world. Researchers have tried to address the challenges of most deficient nutrients like vitamin A, iron, and quality proteins through wheat. The provitamin A content of wheat has been enhanced by expressing bacterial PSY and carotene desaturase genes [CrtB, CrtI (76, 77)]. The iron content in wheat has been enhanced by expression of ferritin gene from soybean (78) and wheat [TaFer1-A (79)]. To increase iron bioavailability phytase activity was increased by the expression of the phytochrome gene [phyA (80)] and phytic acid content has been decreased by silencing of wheat ABCC13 transporter (81). Protein content, especially essential amino acids lysine, methionine, cysteine, and tyrosine contents of wheat grains were enhanced using Amaranthus albumin gene [ama1 (82)]. Wheat has also been targeted to improve the antioxidant activity by expressing maize regulatory genes (C1, B-peru) involved in anthocyanin production (83). To address the challenge of overnutrition and obesity, the content of less digestible and resistant amylose starch has been enhanced by silencing gene encoding SBE [SBEIIa (84)]. ### Transgenic Maize (Zea mays) Maize is one of the important staple crops in developing countries, and it has been addressed for vitamins, minerals, quality protein, and antinutrient components by means of genetic engineering. Maize endosperm has been enriched with provitamin A (carotenoids) by expressing bacterial crtB (85) and multiple (5) carotenogenic genes (86, 194). Vitamin E and its analog are potent antioxidants with implications over human health and many research groups are emphasizing on biofortification of these components in maize crop. Tocotrienol and tocopherol content in maize has been increased by overexpression of homogentisic acid geranylgeranyl transferase [HGGT (87)]. Vitamin C (l-ascorbic acid) a water-soluble antioxidant play roles in cardiovascular function, immune cell development, and iron utilization (88). Its level in corn has been enhanced nearly 100-fold times by recycling oxidized ascorbic acid to reduced form by the expression of dehydroascorbate reductase [DHAR (89)]. On the other hand, Naqvi et al. (90) developed multivitamin corn containing 169-fold the normal amount of beta-carotene, double the normal amount of folate and 6-fold the normal amount of ascorbate by engineering three distinct metabolic pathways. Bioavailability of micronutrients is hindered by antinutrient components. Bioavailability of iron has been increased by expressing soybean ferritin and Aspergillus phytase (91), soybean ferritin (92), Aspergillus niger phyA2 (93), and silencing the expression of ATP-binding cassette transporter and multidrug resistanceassociated protein (94). As a practical example, BVLA4 30101 variety released by Origin Agritech in China has been biofortified for phytate degradation. The major maize seed storage proteins, zeins have poor nutritional quality due to lower content of essential amino acids lysine and tryptophan. In maize essential amino acid content has been targeted with significant achievement. Lysine content in maize has been increased by expression of sb401 from potato (95, 96), single bifunctional expression/silencing transgene cassette (97). Both lysine and tryptophan content have been increased in maize by antisense dsRNA targeting alpha-zeins [both 19- and 22-kDa (98)]. Importance of lysine content in maize is evident from maize varieties rich in lysine viz., Mavrea™YieldGard Maize that has been released by Monsanto in Japan and Mexico; Mavera™ Maize (LY038) by Renessen LLC (Netherland) in Australia, Columbia, Canada, Japan, Mexico, New Zealand, Taiwan, USA The amino acid methionine is a common protein building block that is also important in other cellular processes. Its content has been increased in maize by modifying cis-acting site for Dzs10 (99). Amino acid balance of maize has also been improved by expressing milk protein α-lactalbumin (40). ### Transgenic Barley (Hordeum vulgare) Barley being a model cereal crop has been targeted to improve its micronutrient content. Its zinc content has been improved by overexpression of zinc transporters (100). To increase the bioavailability of iron and zinc, phytase activity has been increased in barely seeds by expression of phytase gene [HvPAPhy\_a (101)]. Essential amino acid lysine has been enhanced in barley by expressing DHPS gene [dapA (102)]. β glucans are dietary fibers and are believed to dramatically reduce the risk of contracting serious human diseases such as cardiovascular disease and type II diabetes (103). Its content has been increased in barley by overexpression of cellulose synthase-like gene [HvCslF (104)]. Resistant starch (amylose only) barley has been produced by the RNAi approach by suppressing all genes coding for SBE [SBE I, SBE IIa, SBE IIb (105)]. Content of health promoting polyunsaturated fatty acids, γ-linolenic acid, and stearidonic acid (STA) has been improved in barley by expressing Δ<sup>6</sup> -desaturase [D6D (106)]. Barley has been targeted to express human lactoferrin gene [HLF (107)]. Apart from this several medicinally and industrially important bioactives including enzymes and antibiotics have been expressed in barley. ### Transgenic Sorghum (Sorghum bicolor) Sorghum is one of the most important staple foods for millions of poor rural people. It has an ability to grow well in harsh environments. It has been targeted to improve provitamin A (beta-carotene) by expressing Homo188-A (108). Content of essential amino acid lysine has been improved in sorghum by the introduction of a high lysine protein [HT12 (109)]. One of the issues with sorghum consumption is that its grains are less digestible than the other major staple crops. Its seed storage proteins, γ-kafirin, is resistant to protease digestion. Digestibility index of transgenic sorghum has been increased by RNAi silencing of the γ-kafirin (110) and combined suppression involving three genes [γ-kafirin-1, γ-kafirin-2, and α-kafirin A1 (111)]. ### TRANSGENIC LEGUMES AND PULSES ### Transgenic Soybean (Glycine max) Soybean is a global source of vegetable oil and high-quality protein. The soybean has been targeted to increase provitamin A (beta-carotene), a monounsaturated ω-9 fatty acid (oleic acid) and seed protein contents by expressing bacterial PSY gene (112). In a different approach provitamin A (Canthaxanthin) was enhanced by expressing bacterial PSY [crtB, crtW, bkt1 (113)]. Kim et al. (114) has demonstrated the production of a high provitamin A (beta-carotene) soybean through overexpression of PSY and carotene desaturase. Another important nutrient vitamin E activity in barley has been enhanced with increased content of δ-tocopherol and decreased γ-tocopherol by coexpressing 2-methyl-6-phytyl benzoquinol methyltransferase genes [At-VTE3; At-VTE4 (115)]. Soybeans contain approximately 40% protein, but they are deficient in one or more of the essential amino acids, especially the sulfur-containing amino acids, cysteine and methionine. The cysteine content of soybean seeds has been increased through overexpression of the sulfur assimilatory enzyme, O-acetylserine sulfhydrylase (116). Similarly, Dinkins et al. (117) increased methionine and cysteine content in soybean by overexpressing the maize zein protein. The methionine content of soybean has been increased by expressing cystathionine γ-synthase (118, 119). Soybean is rich in healthy oil and has approximately 20% oil content. But 7–10% of the oil contains unstable fatty acid α-linolenic acids that contribute to reduced soybean seed oil quality. It results in the formation of undesirable trans-fatty acid as a result of hydrogenation (195). To enhance the agronomic value of soybean seed oil by reducing the levels of α-linolenic acids (18:3), siRNA-mediated gene silencing-based approach has been utilized for silencing of ω-3 FAD3 (120). In another experiment γ-linolenic acid (GLA) and STA (ω-3 fatty acids) content in soybean oil has been increased by expression of Δ<sup>6</sup> -desaturase gene that is responsible for the conversion of linoleic acid and α-linolenic acid to GLA and STA (121). Similarly, STA content has been increased by simultaneous expression of Δ<sup>6</sup> desaturase and Δ15 desaturase (122). Antisense RNA technology has been used to reduce the amount of linoleic acid and palmitic acid and increase the amount of oleic acid by inhibition of expression of Δ12 oleate desaturase [GmFAD2-1b (123)] that converts oleic acid into linoleic acid. Soybean seeds are low in isoflavone content. Consumption of isoflavone is associated with human health benefits such as decreased risk of heart disease, reduced menopausal symptoms, and reduced risk of some hormone-related cancers (196). Isoflavone content has been enhanced in soybean seeds by the combination of maize C1 and R transcription factor-driven gene activation and suppression of a competing pathway (124). Importance of improvement in ω-3 fatty acid content in soybean is evident from the fact that a large number of cultivars with improved oleic, linoleic, and STA have been released by private companies. Transgenic soybean varieties rich in oleic acid viz., G94-1, G94-19, G168 have been released in Australia, Canada, Japan, New Zealand, USA; and Treus™, Plenish™ (DP305423) in Australia, Canada, China, European Union, Japan, Mexico, New Zealand, Philippines, Singapore, South Africa, South Korea, Taiwan, USA; and Treus™ (DP 305423 × GTS 40-3-2) in Argentina, Canada, China, Japan, Mexico, Philippines, South Africa, South Korea, Taiwan by Dupont. The transgenic varieties of soybean rich in oleic acid were released by Monsanto, viz., Vistive Gold™ (MON87705) in Australia, Columbia, Canada, European Union, Indonesia, Japan, Mexico, New Zealand, Philippines, Singapore, South Korea, Taiwan, USA, Vietnam; MON87705 × MON87708 × MON89788 and MON 87705 × MON 87708 × MON 89788 in Canada. The soybean variety rich in oleic acid and linoleic acid was released in the European Union, Mexico, South Korea, and Taiwan. The other varieties rich in STA viz., MON 87769 × MON 89788 were released in Mexico, South Korea, Taiwan and MON87769 released in Australia, Columbia, Canada, European Union, Indonesia, Japan, Mexico, New Zealand, Philippines, South Korea, Taiwan, USA, Vietnam by Monsanto company. ### Transgenic Common Beans (Phaseolus vulgaris) The common bean is among the most important grain legumes used for human consumption. However, although beans are rich in some essential amino acids, e.g., lysine, threonine, valine, isoleucine, and leucine, their nutritional value is limited because of the small amounts of the essential amino acid methionine and cysteine. Common bean methionine content has been increased by the expression of methionine-rich storage albumin from Brazil nut (125). ### Transgenic Lupines (Lupinus angustifolius) Lupine is the major grain legume. The lupine seed protein, in common with the protein of most other grain legumes, is deficient in the sulfur-containing amino acids methionine and cysteine. Its methionine content has been increased by the expression of sunflower seed albumin gene (126). ## TRANSGENIC VEGETABLES ### Transgenic Potato (Solanum tuberosum) Potato is the world's fourth most important source of calories, and it's any nutritional enhancement is of great significance. In potato tuber, provitamin A (carotenoid forms) have been increased by incorporating PSY gene (127) and by simultaneous incorporation of three genes: PSY, phytoene desaturase, and lycopene β-cyclase (128). Beta-carotene content in tubers has been also enhanced by using RNAi to silence the beta-carotene hydroxylase gene (bch), which converts beta-carotene to zeaxanthin (129) and by regulation of beta-carotene synthesis through expression of lycopene β-cyclase [StLCYb (130)]. In another experiment, it has been observed that incorporation of Or gene from orange cauliflower mutant leads to increase in carotenoids along with three additional metabolite intermediates phytoene, phytofluene, and z-carotene (131). Zeaxanthin which is another form of carotenoid has been also increased by expressing zeaxanthin epoxidase genes in transgenic potato tuber (132). The potato has been also targeted for enhancement of vitamin C (ascorbic acid) by overexpressing strawberry GalUR (133). Potato tubers are very poor in essential amino acid, methionine, which has been targeted for its enhancement by coexpressing cystathionine γ-synthase (CgSΔ90) and methionine-rich storage protein (134). Similarly, silencing of StMGL1 (135) and antisense inhibition of threonine synthase (136) led to increase in methionine to isoleucine ratio and methionine content (up to 239-folds) in potato tubers. Methionine content has been also enhanced by overexpressing the gene encoding the seed storage protein from Perilla [PrLeg polypeptide (137)] and cystathionine γ-synthase (CgS) genes (138). Transgenic potatoes expressing Amaranth albumin (ama1) result in an increase in total protein content in tubers along with the significant increase in the concentration of several essential amino acids including methionine (139). High value carbohydrate rich potato tubers has been synthesized by expressing cyclodextrin glycosyltransferases (CGT) gene, which results in the production of multipurpose dietary fiber cyclodextrins from starch (140). Potato tubers have been also focused upon to increase the phenolic acid, and anthocyanins contents by the single-gene overexpression or by simultaneous expression of CHS, chalcone isomerase (CHI), and dihydroflavonol reductase (141). It has been also targeted to improve the content of dietary fiber fructan and inulin (142, 143). Transgenic potato varieties engineered for starch quality, which has reduced amylose and increased amylopectin in starch granules were released by BASF viz., Starch Potato (AM 04—1020) in the USA and Amflora™ (EH 92-527-1) in the European Union. Transgenic potato varieties that limit formation of the reducing sugars through starch degradation have been released in Canada and USA by J. R. Simplot Co. ### Transgenic Sweet Potato (Ipomea batatas) Sweet potato is an alternative source of bioenergy and natural antioxidants. It is rich in various phytochemicals, anthocyanins, vitamin C, carbohydrates, potassium, and dietary fiber (197). Its nutrition properties have been further enhanced by increasing the contents of carotene, lutein, and total carotenoids by overexpressing orange IbOr-Ins gene in white fleshed sweet potato (144). The antioxidant capacity of orange-fleshed sweet potato cultivar has been increased by overexpression of IbMYB1 a key regulator of anthocyanin biosynthesis in the storage roots (145). ### Transgenic Cassava (Manihot esculenta) Cassava is an important staple food crop for millions of poor people worldwide as it is tolerant to different stresses. However, cassava is deficient in several important nutrients like provitamin A, vitamin E, iron, and zinc. Cassava biofortification of provitamin A, iron, and zinc has been carried out to reduce their deficiency among the undernourished communities. Telengech et al. (146) as a part of the BioCassava Plus project developed transgenic cassava that expresses beta-carotene in roots using nptII, crtB, and DXS. Similarly, Welsch et al. (147) showed that the cassava plants overexpressing a PSY transgene produced yellow-fleshed, high-carotenoid roots. Different transgenic cassava varieties biofortified for enhanced levels of iron, beta-carotene, and zinc are under development and field trials in the Biocassava Plus Program targeted at African countries. ### Transgenic Carrot (Daucus carota subsp. sativus) Carrots are one of the most popular vegetables and contain high levels of beta-carotene and vitamins and minerals; however, like many vegetables, these are poor in calcium content (198). Bioavailable calcium content in transgenic carrot has been increased by expressing the Arabidopsis H<sup>+</sup>/Ca2<sup>+</sup> transporter [CAX1 (148, 149)]. ### Transgenic Lettuce (Lactuca sativa) Lettuce is one of the most popular leafy vegetables all around the world. Compared to spinach, the iron content of lettuce is low. The lettuce has been improved for iron content, yield, and growth rate by expressing a soybean ferritin gene (150). ### Transgenic Cauliflower (Brassica oleracea) Cauliflower is a popular vegetable in several parts of the world. It is rich in antioxidant phytonutrients. Its nutritional value has been further enhanced by increasing beta-carotene content in mutant orange cauliflower by the insertion of a copia-like LTR retrotransponson in the Or (151). ## TRANSGENIC OILSEEDS ### Transgenic Linseed (Linum usitatissimum) Linseed edible oil is in demand as a nutritional supplement. Linseed or flax seeds are the richest source of polyunsaturated fatty acids, but linseed oil is highly susceptible to auto-oxidation, which generates toxic derivatives. Genetically modified flax plants with increased antioxidant potential, stable, and healthy oil production has been generated by suppressing CHS gene that resulted in hydrolyzable tannin accumulation (152). Very longchain unsaturated fatty acids (VLCPUFA) are important fatty acids with limited supply due to decrease in marine resources such as fish oils. It can be compensated by implementation of VLCPUFA biosynthesis into oilseed crops (153). VLCPUFA such as arachidonic acid (C20:4 n-6), eicosapentenoic acid (EPA C20:5 n-3), and docosahexenoic acid (DHA C22:5 n-3) are considered to be nutritionally beneficial because of their function as cholesterol-lowering agents (199). Researchers have intended to enhance the accumulation of Δ<sup>6</sup> desaturated C18 fatty acids and C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid by seed-specific expression of cDNAs encoding fatty acyl-desaturases and elongases in linseed (154). Enrichment of carotenoids in flaxseed has been done by the introduction of PSY gene [crtB (155)]. Transgenic linseed rich in essential amino acids viz., CDC Triffid Flax (FP967) has been released by University of Saskatchewan, in Colombia, USA, and Canada. ### Transgenic Canola (Brassica napus) Canola is an important oilseed crop for millions of people around the world. Canola produces edible oil lower in saturated fat and higher in omega-3 fatty acids. To further enhance its health benefits its carotenoid content (mainly alpha and beta-carotenes) has been increased by overexpressing bacterial PSY [crtB (37)]. Higher β-carotenoid content has been achieved by simultaneous expression of PSY, phytoene desaturase, and lycopene cyclase genes (155) and simultaneous expression of seven bacterial genes; idi, crtE, crtB, crtI, crtY, crtW, and crtZ (157). Higher beta-carotene content along with high xanthophylls and lutein contents have been achieved by RNAi silencing of lycopene ε-cyclase [ε-CYC (158)] and DET1 (159). Essential amino acid lysine has been increased in canola by expression of aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS) genes (160). Increase in level of two fatty acids viz., caprylate (8:0) and caprate (10:0) in canola seed oil accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3) has been achieved by overexpression of thioesterase gene [Ch FatB2 (161)]. Canola normally does not have any Δ<sup>6</sup> desaturase activity and thus lack GLA. In order to produce GLA more economically and to make it more readily available transgenic lines rich in GLA has been developed by expression of Δ12 or Δ<sup>6</sup> desaturases genes (162, 163). Phytic acid is known as a food inhibitor, which chelates micronutrient and prevents its bioavailability, as human and other monogastic animals lack the phytase enzyme in their digestive track. Transgenic canola varieties viz., Phytaseed™ Canola (MPS 961-965) engineered for phytase degradation to enhance the availability of phosphorus in canola has been produced and released by BASF in USA. ### Transgenic Mustard (Brassica juncea) Mustard is an economically significant crop and extensively cultivated for oil throughout the world. It has been targeted for improving the nutritionally important unsaturated fatty acids. This has been achieved by the expression of the enzyme Δ<sup>6</sup> FAD3 that led to the production of gamma linoleic acid in the transgenic mustard (164). ## TRANSGENIC FRUITS ### Transgenic Tomato (Solanum lycopersicum) Tomato is one of the most popular fruits, consumed by billions around the world and is an important source of vitamin C, micronutrients, and other phytonutrients. It derives its color from isopernoid lycopene. Isoprenoids are one of the largest classes of natural products with several thousand compounds. In higher plants, isoprenoids have essential roles in membrane structure (sterols), free radical scavenging (carotenoids and tocopherols), redox chemistry (plastoquinone, ubiquinone), defense mechanisms (phytoalexins), and growth regulation (gibberellins, cytokinins, brassinosteroids, and abscisic acid) (200). Several attempts have been made to increase the isoprenoid content in tomato. The sterol content was elevated in tomato by expression of 3-hydroxymethylglutaryl CoA [hmgr-1 (165)]. Tomato phytoene and beta-carotene content has been enhanced by expression of 1-deoxy-d-xylulose-5-phosphate synthase [dxs (165)]. Higher contents of lycopene, betacarotene, and lutein have also been achieved in tomato by the expression of PSY gene [crtB (166)]. Double biofortification of carotenoid and flavonoid contents have also been achieved by RNAi technology by suppressing photomorphogenesis regulatory gene [DET1 (172)]. The beta-carotene content has also been increased by overexpression of lycopene beta-cyclase gene [beta-Lcy (167–169)]. Higher contents beta-carotene as well as its hydroxylation product xanthophylls (beta-cryptoxanthin and zeaxanthin) has been obtained by simultaneous expression of beta-Lcy and beta-carotene hydroxylase [b-Chy (171)]. Total carotenoid and high value astaxanthin content (hydroxylation product of a beta-carotene) have been enhanced in tomato by expression of beta-carotene ketolase and hydroxylase (170). The tomato has been targeted to improve its vitamin C (ascorbic acid) content by overexpressing GDP-mannose 3′,5′-epimerase [SlGME1, SlGME2 (173)], DHAR (174), and coexpression of three genes GDP-mannose pyrophosphorylase, arabinono-1,4-lactone oxidase, and myo-inositol oxygenase 2 (88, 175). Another important nutrient folic acid has been targeted by overexpression of GTPCHI (176) and aminodeoxychorismate synthase (177). Tomato has also been selected to increase antioxidant anthocyanins by expression of CHI (178), transcriptional activators AtMYB75 (179), and expression of two transcription factors, Delila and Rosea1 (201). Other antioxidants like chlorogenic acid have been targeted by gene silencing of HQT (180), transresveratrol by expression of stilbene synthase (181), polyphenolic antioxidants by expression of AtMYB12 (182), and genistin by overexpression of isoflavone synthase (IFS) gene (183). Anthocynin rich blue transgenic tomato has been developed by Norfolk plant sciences. ### Transgenic Apple (Malus domestica) Apple has long been recognized as a great source of antioxidants. Apple has been bioengineered with a stilbene synthase gene from the grapevine (Vitis vinifera L.) thereby leading to synthesis of resveratrol in transgenic apple, thereby, expanding the antioxidant capacity (184). ### Transgenic Banana (Musa acuminata) The banana, a fourth most important food crop of the developing countries, has been predominantly targeted for beta-carotene. This has been achieved by developing transgenic banana (Super Banana) by expressing PSY gene (PSY2a) of Asupina banana, which is naturally high in beta-carotene (185). ### TRANSGENIC FODDER ### Transgenic alfalfa (Medicago sativa) Alfalfa is as an important feed legume crop in many countries. Attempts have been made to improve its nutritional status through enhancement of isoflavonoids, essential amino acids, and improve its digestibility. Isoflavonoids are a predominantly legume-specific subclass of flavonoid secondary metabolites. Transgenic alfalfa has been generated by constitutively expressing IFS that is correlated with its increased isoflavonid composition (186). Alfalfa suffers from a limited level of the sulfur-containing amino acids, methionine, and cysteine. Its methionine content has been increased by the expression of cystathionine γ-synthase [AtCgS (187)]. Improvement in the digestibility of forages has also been an area of interest as it correlates with animal performance. By targeting three specific cytochrome P450 enzymes for antisense downregulation, transgenic alfalfa lines have been generated with low lignin content (188). Alfalfa has also been engineered to increase phytase activity, and thereby enabling its use in animal feeds, including livestock, poultry, and fish feed (189). ### BIOFORTIFICATION THROUGH AGRONOMIC APPROACHES Biofortification through agronomic methods requires physical application of nutrients to temporarily improve the nutritional and health status of crops and consumption of such crops improves the human nutritional status (202). In comparison with inorganic forms of minerals, the organic ones are more available for a man, as they can be absorbed more easily; and are less excreted (203) and their toxicity symptoms are less intensive (DRI 2000). It generally relies on the application of mineral fertilizers and/or increase in their solubilization and/or mobilization from the soil in the edible parts of plants. Macrominerals like nitrogen, phosphorus, and potassium (NPK) make an important contribution to the attainment of higher crop yields (204). Through the application of NPK-containing fertilizers, agricultural productivity increased in many countries of the world in the late 1960s and resulted in Green Revolution and saved them from starvation. In the current scenario, these fertilizers are important and necessary to improve crop yield and save the human population from starvation as low-input agriculture cannot feed the current seven billion world population (205). Microminerals iron, zinc, copper, manganese, I, Se, Mo, Co, and Ni are found in varying degrees in the edible portion of certain plants and are usually absorbed from the soil. Improvement of the soil micronutrient status by their application as fertilizers can contribute to decrease in micronutrient deficiency in humans (206). When crops are grown in soils, where mineral elements become immediately unavailable in the soil and/or not readily translocated to edible tissues targeted application of soluble inorganic fertilizers to the roots or to the leaves are practiced. Agronomic biofortification is simple and inexpensive, but needs special attention in terms of source of nutrient, application method and effects on the environment. These should be applied regularly in every crop season and thus are less cost-effective in some cases. Use of mineral fertilizers is evidently feasible in the developed world, as exemplified by the success of Se fertilization of crops in Finland (207), zinc fertilization in Turkey (208), and I fertilization in irrigation water in China (209). In addition to fertilizers, plant growth-promoting soil microorganisms can be used to enhance the nutrient mobility from soil to edible parts of plants and improve their nutritional status. Soil microorganisms like different species of genera Bacillus, Pseudomonas, Rhizobium, Azotobacter, etc. can also be utilized to increase the phytoavailability of mineral elements (210, 211). The N2-fixing bacteria play important role in increasing crop productivity in nitrogen limited conditions (212). Many crops are associated with mycorrhizal fungi that can release organic acids, siderophores, and enzymes capable of degrading organic compounds and increasing mineral concentrations in edible produce (210, 213). Different crops have been targeted through agronomical biofortification to improve the human nutritional status (Table 3). ### CEREALS ### Rice Agronomic Biofortification Micronutrient biofortification through agronomical practices is an alternative strategy to reduce the iron and zinc deficiency in rice grain. Biofortification of rice plants by foliar spray of iron was an effective way to promote iron concentration in rice grains (214–216). Similarly, fortifying germinating rice plantlets with ferrous sulfate lead to increase iron concentration in germinated brown rice [up to 15.6 times the control (215)]. Foliar application of zinc has been reported as an effective agronomic practice to promote rice grain zinc concentration and zinc bioavailability (216, 218–223). On the other hand, application of zinc to soil as fertilizer in addition to a foliar spray proves to be an important strategy to increase the grain zinc content of rice grown in soils with low background levels of zinc (224). Selenium, which is an essential trace element for human health and proved to be a potent antioxidant, has been also increased by the application of selenate as a foliar spray or as fertilizer in rice (216, 225–230). ### Wheat Agronomic Biofortification Agronomic biofortification has been very efficiently utilized in wheat grain quality improvement. Inclusion of iron in foliar urea fertilizers has been positively correlated with high iron accumulation (231). Application of foliar zinc has reduced human zinc deficiency in regions with potentially zinc-deficient soil and also improved its bioavailability by reducing antinutrient factors like phytic acid (233). Due to significant effects of zinc fertilizers on grain yield, the total amount of zinc-containing NPK fertilizers increased from 0 in 1994 to a record level of 400,000 t per annum in 10–15 years in Turkey. Use of zinc-containing fertilizers increased zinc concentration in grain, and obviously contributed to human nutrition and health in Turkey, especially in rural areas, where wheat provided more than 50% of the daily calorie intake (206). Agronomic biofortification of Se in wheat has been adopted with success in Finland (207). Compound fertilizers supplemented Table 3 \| Tabulation of crops, nutrients, research status, and concerned publications on biofortification through agronomic approaches. (Continued) #### TABLE 3 \| Continued Physical application of nutrients, growth-promoting soil microorganisms, N2-fixing bacteria and mycorrhizal fungi are utilized to increase the mineral concentration in edible produce. with Se were utilized since 1984, and it resulted in an increase in human serum selenium. Apart from chemical and organic fertilizers, researchers have also investigated the role of biofertilizers in promoting the yield of grains. Mycorrhizal fungi along with fertilizers are extensively being used for biofortification (234). Iron biofortification of wheat grains has been accomplished through integrated use of organic and chemical fertilizers and zinc biofortification by using Bacillus aryabhattai (235, 236). ### Maize Agronomic Biofortification Among micronutrients, zinc is required for obtaining nutrientenriched grain and optimum yield in maize. For achieving this, various zinc fertilizer treatments and foliar applications have been carried out in maize crop (237, 239–241). Plant growthpromoting rhizobacteria have led to nutrient enrichment in the plants and have been included in agronomic approaches to develop effective biofortification strategies for the staple crops. One of the effective examples is the maize crop with increased zinc content (242). The Selenium (Se) importance in human and animal health has been known worldwide, and it has also been increased by applying fertilization as an effective agronomic biofortification strategy (226). ### Barley Agronomic Biofortification The micronutrient profile of barley has been improved by the application of various organic and inorganic biofertilizers. The concentration of zinc and iron in grains has been enhanced by the application of biofertilizers along with inorganic fertilizers and vermicompost (243). ### Sorghum Agronomic Biofortification Sorghum is cultivated worldwide for grain and fodder. This crop often suffers from the challenge of growing in nutrient poor and contaminated soil. Its nutrient profile has been promoted by the application of fertilizers (both organic and inorganic) that have an additive effect on the yield. Researchers have intended to improve the nutrient uptake and alter the metabolic profile of sorghum by using the combination of plant growth-promoting bacteria and arbuscular mycorrhizal fungi (AMF) (244, 245). Also, the inoculation of Azospirillum alone and in combination with phosphate-solubilizing bacteria increased sorghum grain yield and protein content by improving the status of phosphorous and nitrogen in the soil (246). ## LEGUMES ### Soybean Agronomic Biofortification Selenium-enriched soybean has been produced by the foliar application of selenium complex salts as fertilizers (247). ### Chickpea Agronomic Biofortification Chickpea has been targeted for the mineral deficiencies, especially the mineral iron, zinc, calcium, copper, manganese, and Mg by using plant growth-promoting actinobacteria (248). Chickpea biofortification for iron and zinc has been addressed by using AMF (249). Similarly, zinc and Se have been fortified in chickpea by foliar spray of respective minerals (250, 251). ### Pea Agronomic Biofortification Field peas are the second largest legume crop worldwide, also known for their high protein content and its enrichment for zinc has been obtained with foliar zinc applications alone or in combination with soil zinc applications (252). ### Common Bean Agronomic Biofortification A common bean is an herbaceous annual plant grown for edible dry grain. Beans are a good vehicle for zinc biofortification and have been enriched with zinc by the application of foliar zinc fertilizer (223, 253). Furthermore, it has been studied that administration of organic and chemical fertilizers stimulated the uptake of N, P, K, copper, manganese, and zinc in common bean (254). ## OILSEEDS ### Canola Agronomic Biofortification Canola supplemented with plant growth-promoting rhizobacteria viz. Azospirillum brasilense, Azotobacter vinelandii along with chemical fertilizers resulted in increased protein, oleic acid, and linoleic acid content in the seed which indicated that rhizobacteria are highly effective in improving yield and nutritive value of canola oil (263). ### Mustard Agronomic Biofortification Mustard has been targeted for Se enhancement. Plant uptake of Se as selenate has been enhanced by rhizosphere bacteria from a seleniferous area (255). ## VEGETABLES ### Potato Agronomic Biofortification Field experiments were undertaken to increase zinc concentrations in potato tubers (both flesh and skin of tubers) using foliar zinc fertilizers, which significantly increased tuber zinc concentrations. It was also found that zinc oxide and zinc sulfate were more effective than zinc nitrate as foliar fertilizers for increasing tuber zinc concentrations while maintaining yields (264). Increase in Se content of potato tubers has been reported after foliar application of selenium, selenite, and selenate to potato (256, 257). Foliar application of selenium with humic acids was proven to be a good way to increase the selenium content of potatoes (256). ### Sweet Potato Agronomic Biofortification Increase in beta-carotene in orange-fleshed sweet potato has been observed with irrigation and chemical fertilizer treatments (258). ## Carrot Agronomic Biofortification Carrot leaves and storage roots have been supplemented with I and Se by application of both as fertilizers. It has been reported that consumption of 100 g fresh weight of carrots fertilized with I and Se (KICNa2SeO3, KIO3CNa2SeO3) can supply 100% of the recommended daily allowance (259). ## Lettuce Agronomic Biofortification Lettuce I and Se biofortification have been achieved by the application of KIO3 and Na2SeO4 as foliar spray and nutrient medium (260). Lettuce Se biofortification in the leaves has been carried out with good results after soil agronomic biofortification with an inorganic form of selenium (261). ## FRUIT ### Tomato Agronomic Biofortification Studies have concluded that a tomato is an excellent crop for iodine biofortification programs when treated with iron fertilizers (262). ### Biofortification through Conventional Breeding—Most Trusted Approach Biofortification through conventional breeding in the most accepted method of biofortification. It offers a sustainable, cost-effective alternative to transgenic- and agronomic-based strategies. Sufficient genotypic variation in the trait of interest is necessary for conventional breeding to be feasible. Breeding programs can utilize this variation to improve the levels of minerals and vitamins in crops. In conventional plant breeding, parent lines with high nutrients are crossed with recipient line with desirable agronomic traits over several generations to produce plants with desired nutrient and agronomic traits. However, breeding strategies have to sometimes rely on the limited genetic variation present in the gene pool. In some cases, this can be overcome by crossing to distant relatives and thus moving the trait slowly into the commercial cultivars. Alternatively, new traits can be introduced directly into commercial varieties by mutagenesis. Because this approach is likely to be the most expedient method to improve plants, several international organizations have initiated programs to improve the nutritional content of crops through breeding programs. The Health grain Project (2005–2010) involving 44 partners from 15 countries and over £10 million was carried out in the European Union to develop health promoting and safe cereal foods and ingredients of high eating quality. It has since developed into the Healthgrain forum with a wide range of participants from academia and industry. More than 100 publications have reported bioactive compounds in whole-grain cereals, genetic variation, heritability, and effect on reducing risks of many lifestyle-related diseases (265–267). The CGIAR along with the International Center for Tropical Agriculture (CIAT) and the International Food Policy Research Institute have launched the HarvestPlus program to breed biofortified staple food crops. HarvestPlus is investing heavily to boost three key nutrients-vitamin A, iron, and zinc and is targeting the staple crops, wheat, rice, maize, cassava, pearl millet, beans, and sweet potato in Asia and Africa (268). It is directed to produce staple food crops with enhanced levels of bioavailable essential minerals and vitamins that will have measurable impact on improving the micronutrient status of target populations, primarily resource-poor people in the developing world. The Biocassava Plus program had been initiated to improve the nutrition status of cassava crop. Due to better acceptability, large numbers of crops have been targeted for biofortification through crop breeding (Table 4). ## CEREALS ### Rice Breeding Rice is greatly emphasized for micronutrient enhancement. It is one of the most consumed staple food crop and its biofortification can have a significant effect on malnutrition challenge. The milled rice is poor source of minerals. Different old rice varieties with high iron and zinc content in grain have been screened and the higher mineral trait has been combined with improved agronomic traits by breeding methods. The world's first zinc enriched rice varieties developed by HarvestPlus were released in 2013 by Table 4 \| Tabulation of crops, nutrients, research status, and concerned publications on biofortification through breeding. (Continued) (Continued) #### TABLE 4 \| Continued Breeding is so far the best method for crop biofortification. Large number of biofortified cultivars have been released by this approach that are helping in addressing the challenge of micronutrient malnutrition prevalent in the developing countries. Released varieties and their country of release have been bold faced. the Bangladesh Rice Research Institute (BRRIdhan 62, BRRIdhan 72, and BRRIdhan 64), which is claimed to contain 20–22 ppm zinc in brown rice. In India and Philippines, an improved line (IR68144-3B-2-2-3) was identified in a cross between a highyielding variety (IR72) and a tall, traditional variety (Zawa Bonday) with a high concentration of grain iron [about 21 ppm in brown rice (269)]. Similarly, Jalmagna, a traditional variety which had almost double the iron concentration of common rice variety and zinc concentration, nearly 40% more than that of common rice variety has been identified for further breeding programs to improve iron and zinc concentration (269). ### Wheat Breeding Wheat as a staple crop is the first and foremost target for biofortification. Wide variation in grain iron and zinc concentrations in wheat and its closely related wild species has been observed that it can be exploited for improvement of modern elite cultivars (270, 272, 297). Utilizing this variation HarvestPlus has released several varieties of wheat with 4–10 ppm higher zinc content. Six varieties of high zinc wheat (BHU 1, BHU 3, BHU 5, BHU 6, BHU 7, and BHU 18) were released in India in 2014 followed by the release of four varieties in Pakistan in 2015 (NR 419, 42, 421, and Zincol). Two varieties BHU 1 and BHU 6 have high yield, disease resistance in addition to high zinc. Recently, variety with high zinc (PBW1Zn) has been released by Punjab Agricultural University, India. Another variety with high zinc and iron content (WB2) has been developed and released by Indian Institute of Wheat and Barley Research, India. Apart from releasing cultivars, several researchers have reported an increase in the zinc and iron content of wheat by plant breeding (208, 270–272). Provitamin A has been another important nutrient targeted for biofortification through breeding. High provitamin A durum wheat variety (HI 8627) has been released by the Indian Agricultural Research Institute (IARI), India in 2005. Several new cultivars have been released after that with the improved beta-carotene content. Yellow pigment content (YPC; carotenoids mainly xanthophyll lutein) in durum wheat is an important quality trait and an antioxidant. A large number of recent durum wheat varieties released in different countries in the past decade show significantly higher YPC than the old varieties released before the 1970s [(273, 274) and others]. Improvement of antioxidant properties contributed by anthocyanins had also been an area of significant research in wheat. Colored wheat (black, blue, and purple) trait has been used in several breeding programs in different countries. Blackgrained wheat cultivar has been released in China after more than 20 years running effort in breeding and has been reported to be high in protein content and selenium (298). The purple wheat cultivar Indigo has been released in Austria in 2006 (299). The purple wheat cultivar PS Karkulka has been registered in Slovakia in 2014. Purple, blue, and black white lines have been developed and registered in India in 2017 (275). The importance of colored wheat can be adjudged from the patent on functional foods from colored wheat in China (CN102217664 B). Apart from this several researchers have worked on different aspects of colored wheat [reviewed in Ref. (276, 277)]. ### Maize Breeding Maize is a cash crop grown for animal feed, industrial purposes (source of sugar, oil, starch, and ethanol) and for use for human consumption. The vast genetic diversity of maize has been the basis for the breeding programs that have generated much of the higher yielding maize used worldwide. Scientists have discovered varieties that have naturally high levels of provitamin A. HarvestPlus is using these lines to breed high-yielding varieties of biofortified maize with higher levels of provitamin A to combat vitamin A deficiency. The provitamin A maize is one of the significant achievements in the field of biofortification. Biofortified orange maize varieties have been grown commercially in Zambia (GV662A, GV664A, and GV665A), Nigeria {Ife maizehyb-3, Ife maizehyb-4, Sammaz 38 (OPV), Sammaz 39 (OPV)} and Ghana {CSIR-CRI Honampa (OPV)} since 2013 (300). Malawi, Zimbabwe (ZS242) and Tanzania have also released biofortified orange maize recently (301). As a positive effect an increase in pupillary response was observed among Zambian children consuming vitamin A biofortified maize (301). Breeders have evaluated antioxidants like tocochromanols, oryzanol, and phenolic compounds in proVA biofortified maize (279). Another significant achievement in the field of maize biofortification is quality protein maize (QPM). Maize breeders have developed QPM with high essential amino acids lysine and tryptophan by incorporating opaque-2 (o2) mutant gene from naturally occurring maize into the maize cultivars. International Maize and Wheat Improvement Center (CIMMYT) has released such hybrid varieties in India (CML176, CML176 × CML186, HQPM4, HQPM-7, VivekQPM-9, HQPM-5, HQPM-1, FQH-4567), China (CML140, CML194, P70), Vietnam (CML161 × CML165), Mexico (CML142 × CML176, CML142 × CML150, CML176, CML170, CML186 × CML149, CML176 × CML186), South Africa (QS-7705), Ghana (GH-132-28), Guinea (Obatampa), Uganda (Obangaina), Benin (Obangaina), Mozambique (Susuma), Brazil (BR-451, BR-473), Venezuela (FONAIAP), Peru (INIA), Colombia (ICA), Honduras (HQ-31), El Salvador (HQ-61), Guatemala (HB-Proticta), and Nicaragua (NB-Nutrinta, HQ INTA-993). For QPM maize breeders, Surinder Vasal and Evangelina Villegas won 2000 world food prize. Maize has also been inbred by recurrent selection scheme, to increase the carotenoids (278) alone or in combination of vitamin E and phenolics (279) and antioxidant power (280). Attempts have been made to increase its vitamin E content (281). ## Sorghum Breeding The prospects of breeding for micronutrients and beta-carotene rich sorghums have been discussed by Reddy et al. (282). Sorghum varieties have been screened for high minerals, protein (302), lutein, zeaxanthin, and beta-carotene contents (303). Sorghum germplasm has shown large variability and genetic heritability for iron and zinc content (304). Biofortified iron rich sorghum lines (ICSR 14001, ICSH 14002) and hybrids (ICSA 661 × ICSR 196, ICSA 318 × ICSR 94, ICSA 336 × IS 3760) have been bred by ICRISAT and released in India. New nutritionally high (Fe) sorghum varieties (12KNICSV-22 and 12KNICSV-188) have been released in Nigeria that may boost the malnourished populations, especially children in Nigeria. One of the new varieties (12KNICSV-188) has iron content three times higher than typically grown sorghum. These new varieties involved crossing local Nigerian germplasm with improved lines from ICRISAT (Mali). ### Millets Breeding Pearl millet is the cheapest source of iron and zinc (305) and large variation has been seen in its germplasm for these micronutrients (283). In India, biofortified (iron and zinc) pearl millet variety "Dhanashakti" and a hybrid ICMH 1201 (Shakti-1201) has been released by ICRISAT, HarvestPlus in 2014. Besides that, two varieties, ICMH 1202 (Nirmal-7) and ICMH 1301, are currently undergoing advanced farm trials. Various well-adapted commercial varieties, their progenies, and hybrids containing high content of iron and zinc in grain have been reported (283, 284). ### LEGUMES AND PULSES ### Lentil Breeding Lentil, a key pulse in many dryland countries and has easy to cook properties. It has been directed by ICARDA, HarvestPlus for biofortification of iron and zinc with the help of breeding process using genetic diversity stored in gene banks. Research findings have shown that there is a positive correlation of iron and zinc synthesis with protein synthesis, therefore lentil varieties with higher iron, zinc, and protein content can be developed together [ICARDA, HarvestPlus (306)]. High iron and zinc lentil varieties, five in Bangladesh (Barimasur-4, Barimasur-5, Barimasur-6, Barimasur-7, and Barimasur-8), seven in Nepal (ILL 7723, Khajurah-1, Khajurah-2, Shital, Sisir Shekhar, Simal), two in India (L4704, Pusa Vaibhav), one in Ethiopia (Alemaya), and two in Syria (Idlib-2, Idlib-3) has been released by ICARDA, HarvestPlus biofortification program till date. Lentil varieties have been screened for variation in Se content (307). ## Cow Pea Breeding Cow pea which is also known as poor man meat, rich in protein content has been biofortified for iron content by means of breeding methods. Pant Lobia-1 (2008), Pant Lobia-2 (2010), Pant Lobia-3 (2013), and Pant Lobia-4 (2014) varieties with increased iron content have been released by GB Pant University, Pantnagar, India in collaboration to HarvestPlus. ## Bean Breeding Studies till date suggest that the iron content of the common bean (P. vulgaris) could be increased by 60–80%, while zinc content would be more modest, perhaps around 50%. High heritability has been observed in iron and zinc content in common bean (285, 287, 308). Genes associated with zinc accumulation have been identified in navy bean (286). HarvestPlus is working in this direction and promoting iron biofortified beans in several developing countries. They have released 10 Fe-biofortified common bean varieties in Rwanda (RWR 2245, RWR 2154, MAC 42, MAC 44, CAB 2, RWV 1129, RWV 3006, RWV 3316, RWV 3317, and RWV 2887). HarvestPlus also released ten biofortified iron bean varieties in the Democratic Republic of Congo, i.e., COD MLB 001, COD MLB 032, HM 21-7, RWR 2245, PVA 1438, COD MLV 059, VCB 81013, Nain de Kyondo, Cuarentino, Namulenga. ## VEGETABLES ### Potato Breeding Potato tubers are the richest sources of antioxidants in human diet. The natural variation of cultivated potato germplasm containing red and purple pigment could possibly represent the contribution of the potatoes to the portion of antioxidants in human nutrition. Therefore, effort of breeders focuses on the breeding of such variants (288). Furthermore, vast genetic variation for micronutrients (291) exists in potato that can be exploited for breeding to further increase iron and zinc levels in human diets (290). A genetically diverse sample of potato cultivars native to the Andes of South America has been obtained from a collection of nearly 1,000 genotypes and evaluated as a source of antioxidants and minerals (copper, iron, manganese, and zinc) (289, 292). International potato center (CIP) and HarvestPlus have developed high iron and zinc advanced breeding material after crossing diploid Andean landrace potatoes with high zinc and iron with disease resistant tetraploid clones. The main target countries for biofortified potato are Rwanda and Ethiopia. National Institute for Agrarian Innovation's (INIA) Potato Program has developed the INIA 321 Kawsay variety in Peru that has a high content of iron and zinc. ### Sweet Potato Breeding Developing countries are growing 95% of the world's sweet potato crop, where malnutrition is the biggest problem. The sweet potato has been targeted for improvement in vitamin A. HarvestPlus and International Potato Centre (CIP) have developed and released several varieties of orange sweet potato with high vitamin A. Six varieties have been released in Uganda (Ejumula, Kakamega, Vita, Kabode, Naspot 12O, and Naspot 13O) and three in Zambia (Twatasha, Kokota, and Chiwoko). Zambia Agriculture Research Institute has successfully completed the development of 15 new varieties of vitamin A fortified sweet potatoes. The HarvestPlus orange sweet potato consumption had a significant effect on household food and nutritional security in Sub Saharan Africa, and for this contribution; they have been recently honored with World Food Prize-2016. Furthermore, researchers have identified several sweet potato genotypes that completely lack or have only traces of β-amylase in their storage roots. Such verities could facilitate the breeding of sweet potato for low β-amylase content which can be potentially used for processing and as a staple food (293). ### Cauliflower Breeding Brassica oleracea including cauliflower gene pool has been screened for genetic variation of zinc concentration and sufficient natural variation has been identified (309). The provitamin A (beta-carotene) rich orange colored cauliflower variety (Pusa BetaKesari; 800–1,000 μg/100g) has been released by the Indian Agricultural Research Institute (IARI). Now numbers of colored cauliflower verities are known at world level, having orange and purple color rich in beta-carotene and anthocyanin, respectively. Colored cauliflower varieties, Purple Graffiti and Orange Cheddar, have been developed by Cornell University, USA. ### Cassava Breeding Cassava is a staple vegetable root crop in developing countries, especially in Africa, Latin America, and the Caribbean. In the African continent, it has been targeted for alleviation in provitamin A (beta-carotene) by HarvestPlus in collaboration with International Institute of Tropical Agriculture. Under these collaborations, they have released six vitamin A fortified varieties in Nigeria (2011; TMS 01/1368—UMUCASS 36, TMS 01/1412— UMUCASS 37 and 2014; TMS 01/1371—UMUCASS 38 and NR 07/0220—UMUCASS 44, TMS 07/0593—UMUCASS 45, and TMS 07/539—UMUCASS 46) and one in DRC-Democratic Republic of Congo [Kindisa (TMS 2001/1661)]. Cassava also has a wide range of genotype differences for total carotene, proteins, and minerals (iron and zinc) which has led to the development of improved nutritive value cassava crop (294, 295). ### FRUITS ### Tomato Breeding Tomato is a highly valuable crop and an important source of vitamin A and C. Genetically diverse wild population of tomato has been investigated intensively for specific traits and exploited in tomato breeding (310). Anthocyanin biofortified tomato "Sun Black" with deep purple fruit pigmentation due to high anthocyanin content in the peel has been developed by conventional breeding approach (296). Another variety "Black Galaxy" generated by similar approach has been reported from Israel. ### Banana Breeding Breeding banana is difficult and expensive, as commercial varieties are sterile triploids (3×) and also a high degree of cross incompatibility can exist among the fertile groups. For combating this problem, large scale screening of several banana germplasm for the identification of high levels of provitamin A has been carried out in the Democratic Republic of Congo (DRC) and Burundi by Biodiversity International (BI) in collaboration with HarvestPlus. In this program, they released five varieties (Apantu, Bira, Pelipita, Lai, and To'o) rich in provitamin A in Eastern DRC and Burundi. ### Mango Breeding Mango offers a natural source of beta-carotene, vitamin C, and valuable antioxidants but their nutrient levels vary with mango variety. It has been observed that most of the mango varieties provide more than recommended daily value of vitamin C and beta-carotene. Mango also contains a variety of phenolics like ellagic acid, gallotannin, and mangiferin (311). The Mexicangrown Ataulfo variety ranked highest in both vitamin C (ascorbic acid) and beta-carotene (USDA's Agricultural Research Service). In India, IARI introduced many varieties with enhanced nutritional and agronomical important characters. ### Grape Breeding Grapes have high mineral content, including high vitamins C and K, and are a natural source of antioxidants and other polyphenols, and offer a variety of additional health benefits. Phenolic compounds and antioxidant properties of different grape cultivars grown in China have been assessed (312). The Indian Agricultural Institute has released an improved variety, i.e., Pusa Navrang which contains higher amount of total soluble solids (carbohydrates, organic acids, proteins, fats, and minerals) and antioxidants. ### LIMITATIONS OF BIOFORTIFICATION ### Limitations in Agronomic Biofortification Application of fertilizers fortified with micronutrients is the simplest method among all biofortification methods. But the success of agronomical biofortification is highly variable due to the differences in mineral mobility, mineral accumulation among plant species, soil compositions in the specific geographical location of each crop (313). For example, a study involving diverse rice genotypes indicated that, in the phosphate deficient soils due to reduction in the root biomass, differences in the phosphate uptake among the genotypes were as high as 20-fold (314). Soil composition analysis has indicated that almost 1/2 of the agricultural soils of India, 1/3 of China, 14 Mha of Turkey, 8 Mha of Australia are zinc deficient (315). Agronomic biofortification is less cost-effective and labor intensive as it demands continuous inputs, through the application of micronutrient to the soil or plant regularly. Furthermore, it is not always possible to target the micronutrient into edible plant parts like seed or fruit and can sometimes result in the accumulation of desired nutrients in the leaves or other non-edible portions of plants; therefore, this technique is only successful in certain minerals and specific plant species. For instance, higher zinc efficiency in cereals grown in zinc deficient soils in Turkey was associated with higher uptake of zinc from the soil, but not with increased accumulation of zinc in the grain (208). Furthermore, mineral bioavailability hindered by antinutrient compound like phytic acid is another major challenge (316). In addition, the biggest of all constraints is that the fertilizers accumulation in soil and water poses adverse environmental effects (317). ### Limitations in Conventional Breeding Methods The design of conventional plant breeding programs to improve micronutrient content has proved to be successful and is a sustainable and cost-effective solution in the long run; however, there are limitations with respect to the amount of genetic variability for the micronutrients in the plant gene pool and the time needed to generate cultivars with the desired trait(s). In some cases, this can be overcome by crossing to distant relatives and thus introgressing traits into commercial cultivars, but in many occasions, it would be impossible to breed for a specific trait using conventional means, and the timescale and effort involved may be quite unrealistic, e.g., improving Se concentration in wheat grains (318) and improvement of oleic, linoleic, and linolenic fatty acid content in soybean (319). In general, improvement in oil quality has been targeted with better results with transgenic-based approach (Figure 3B) due to limited variability, heritability, and linkage drag. ### Limitations in Transgenic Methods Transgenic crops overcome the limitation of restricted genetic variation among plants as in the case of conventional breeding but the major limitation of this method is its low acceptance among masses. It is very important that the biofortified crops be readily adapted by farmers and community in significant enough numbers to improve the general nutritional health of a given community (320). Another limitation is that different countries have adopted different regulatory processes for the acceptance and commercialization of these transgenic crops. Regrettably, the current political and economic landscape is not receptive to this technology (321). Furthermore, these regulatory processes are very expensive and time consuming (322). Let us take the example of Bt Brinjal. It has been initially developed by Mahyco, an Indian seed company. Unfortunately, it was not released in Indian because some of the scientists, farmers, and anti-GMO activists, raised concerns and a moratorium on its release was imposed, until further tests were conducted. However, four varieties of Bt Brinjal were given approval for commercial release in Bangladesh in 2013–2014. Although the research efforts devoted to the transgenic-based approach are quite higher compared with breeding based, its success rate in terms of cultivar release in very low (Figure 3A) due to time required from target trait and gene identification, modification, expression, and assessment of agronomical traits to understanding the possible effect on other life forms. For example, after 8 years project, the scientific details of the Golden rice were first published in Science in 2000 (41), and since then different groups, including International Rice Research Institute scientists are working on it, but Golden Rice is still not ready for farmers due to issues with its yield. Its dissemination is also being held back due to inability to get approval from Governments. ### Other Limitations The postharvest processing of each crop must be considered to optimize biofortification strategies. For example, the seeds of many cereals are often consumed after milling or polishing. Although the concentrations of some essential mineral elements, such as Se and S, are highest in the embryo, others, such as iron, zinc, and copper, are highest in the bran (269, 317). Milling or polishing cereal seeds can, therefore, remove large quantities of minerals from the diet; the extent of these losses is genotype dependent (269). In addition, the presence of certain antinutrients in crops reduces the bioavailability of certain nutrients in crops. For examples, antinutrients like phytate, tannins, oxalate, fiber, and hemaglutinins reduce the bioavailability of minerals in human gut (20, 101). Furthermore, in the context of global environmental change, approaches for improving food production, improvements in a crop's ability to maintain yields with lower water supply and quality will be critical. In addition, numerous genes are involved in controlling the amount of a mineral element that is absorbed by roots, translocated to shoot, remobilized from vegetative tissues, and deposited in edible portions of seeds and grains in forms that are utilizable in persons consuming the crop (323, 324). Considerations must also include the micronutrient concentrations in the edible portions of crops, and the amount of nutrients that can be absorbed by the consumer, after processing and cooking (325). ## CONCLUSION It is well established that biofortification is a promising, cost-effective, agricultural strategy for improving the nutritional status of malnourished populations throughout the world. Biofortification strategies based on crop breeding, targeted genetic manipulation, and/or the application of mineral fertilizers hold great potential for addressing mineral malnutrition in humans. The generation of biofortified food crops with improved nutrient contents such as increases in iron, zinc, Se, and provitamin A content are providing sufficient levels of these and other such micronutrients that are frequently lacking in the diets of the developing and developed world. International initiatives, such as the HarvestPlus program and national initiatives, are acting as pillars to achieve these targets. These efforts have delivered crops with the potential to increase both the amounts and bioavailability of essential mineral elements in human diets, especially in staple cereal crops like ### REFERENCES wheat, maize, cassava, beans, sweet potatoes, and millets. But biofortification of crops is a challenging endeavor. To achieve this, collaboration between plant breeders, nutrition scientists, genetic engineers, and molecular biologists is essential. Traditional breeding approaches are finding widespread and easy acceptance and have been used to enhance the nutritional qualities of foods. Although a greater emphasis is being laid on transgenic means success rates of breeding based approaches are much higher as transgenically fortified crop plants have to face hurdles due to acceptance constraints among consumers and different expensive and time consuming regulatory approval processes, adopted by different countries. Besides these challenges, biofortified crops hold a very bright future as these have the potential to remove micronutrient malnutrition among billions of poor people, especially in the developing countries. ### AUTHOR CONTRIBUTIONS MG and NG built the layout of the article, collected literature, and wrote the article. SS and PK collected literature and helped in manuscript writing. AK and VC edited it. PA assisted in reference management. ### ACKNOWLEDGMENTS This work was supported by the National Agri-Food Biotechnology Institute Core grant for improvement of nutrition and processing quality, for which the authors are deeply indebted. produces amylose-only starch granules. BMC Plant Biol (2012) 12(1):223. doi:10.1186/1471-2229-12-223 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Garg, Sharma, Sharma, Kapoor, Kumar, Chunduri and Arora. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Phosphorus Alters Starch Morphology and Gene Expression Related to Starch Biosynthesis and Degradation in Wheat Grain Runqi Zhang† , Cheng Li † , Kaiyong Fu, Chao Li and Chunyan Li\* Xinjiang Production and Construction Group, The Key Laboratory of Oasis Eco-Agriculture, College of Agriculture, Shihezi University, Shihezi, China ### Edited by: Huixia Shou, Zhejiang University, China ### Reviewed by: Miroslav Nikolic, University of Belgrade, Serbia Rumen Ivanov, Heinrich Heine Universität Düsseldorf, Germany \Correspondence: Chunyan Li [email protected] † These authors have contributed equally to this work. ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 01 July 2017 Accepted: 22 December 2017 Published: 12 January 2018 ### Citation: Zhang R, Li C, Fu K, Li C and Li C (2018) Phosphorus Alters Starch Morphology and Gene Expression Related to Starch Biosynthesis and Degradation in Wheat Grain. Front. Plant Sci. 8:2252. doi: 10.3389/fpls.2017.02252 Phosphorus is an essential plant macronutrient which profoundly affects the yield and quality of wheat starch. In this study, scanning electron microscopy showed that P fertilizer amount (0, 46, and 92 kg P ha−<sup>1</sup> ) had no significant effect on the shape of starch granules in wheat (cv. Xindong 20) grain. However, confocal laser scanning microscopy with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde and methanolic merbromin stains indicated that P amount influenced the microstructure of the starch granules. Starch granules from the 46 kg P ha−<sup>1</sup> treatment released significantly more reducing sugars than those from the 0 and 92 kg P ha−<sup>1</sup> treatments during digestion with alpha-amylase and amyloglucosidase digestion. Phosphorus application (especially the 46 kg P ha−<sup>1</sup> treatments) significantly increased the relative expression of genes related to starch synthesis (especially during early to mid-grain filling) and starch degradation (especially during mid- and late grain filling). Phosphorus application also increased the transcript abundance of amylase genes at the periphery of the endosperm. We propose that P application, especially the 46 kg P ha−<sup>1</sup> treatment, enhanced channels in wheat starch granules. These channels facilitated the transport of substances required for starch biosynthesis, thus increasing starch accumulation in wheat endosperm. These results provide insight into the potential mechanisms through which P influences the microstructure and biosynthesis of wheat starch. ### Keywords: biosynthesis, degradation, phosphorus, starch, wheat ### INTRODUCTION Phosphorus (P) is one of the main limiting factors for plant growth in natural ecosystems. The application of P fertilizer is often essential for crop production (Buchanan et al., 2004). Plants generally take up soil P in its inorganic forms. However, 50–80% of the total P in agricultural soils exists as organic phosphate, which is biologically unavailable (Wang et al., 2009). Wheat is grown under P-poor as well as P-rich conditions. In their efforts to maximize field, farmers often over apply P fertilizer. The fertilization rates in some areas are several times greater than the amount required by wheat. This can create serious environmental problems (Tang et al., 2008; Wang et al., 2017). It should also be noted that rock phosphate is a non-renewable resource that is being depleted (Wang et al., 2017). More understanding is needed about how P affects wheat yield and quality. The main component of wheat endosperm is starch, which accounts for ∼70% of grain dry weight. Many enzymes are involved in starch biosynthesis including granule-bound starch synthase (GBSS), adenosine diphosphate glucose pyrophosphorylase (AGPase), starch branching enzyme (SBE), starch debranching enzyme (DBE), and soluble starch synthase (SSS). The AGPase is activated by 3-phosphoglycerate and is inhibited by inorganic P in leaves. The activity of AGPase can be changed by altering the ratio of 3-phosphoglycerate and inorganic P, which in turn regulates starch synthesis (MacDonald and Strobel, 1970). Starch molecules are deposited as semi-crystalline structures in starch granules (Tang et al., 2006). Wheat starch can be classified as either large A-type granules (>10µm diam.) or small B-type granules (<10µm diam.). The two types of granules are differentiated by their morphological and chemical characteristics (Yu et al., 2015). Pores and radial, tube-like channels have been observed at the surfaces of starch granules in wheat (Kim and Huber, 2008), corn, and sorghum (Huber and Bemiller, 2000). It has been hypothesized that the pores are not just surface features but might be openings to channels that provide access to the interior of starch granules (Huber and Bemiller, 2000). Starch granule architecture suggests that the pores and channels may be loosely assembled zones in "defective" blocklets (Tang et al., 2006). Pores and channels within starch granules have important influence on granule accessibility to reagents. This means that pores and channels can influence the reactivity of starch when it is chemically modified for specific purposes in industry (Huber and BeMiller, 1997; Han et al., 2005). Enzymatic digestion is also greatly influenced by pores and channels within native starch granules. Maize starch, which contains pores and channels, is more susceptible to enzymatic hydrolysis than potato starch (Han et al., 2005), which does not have pores (Fannon et al., 1992a). It is possible that starch granule channels can be manipulated to improve digestibility or alter the chemical characteristics of starch (Han et al., 2005). However, much more information is needed about these channels and their biological origin. Environmental conditions such as high temperature (Li et al., 2017) and drought stress (Li et al., 2015) can alter the pits and channels in wheat starch granules. Commuri and Jones (1999) postulated that pitting is induced by imbalances in starch hydrolase and synthase, resulting in premature autolysis. Based on the protein constituents of channels in normal maize starch granules, it has been proposed that the channels are remnants of amyloplast microtubules and may facilitate starch polymer and granule biosynthesis (Fannon et al., 2004; Benmoussa et al., 2010). Previous research in our laboratory indicated that the P fertilizer can also significant affect the characteristics of wheat starch, especially the presence of "pin holes" within starch granules (Li et al., 2013). The objective of this study was to increase understanding about the influence of P fertilizer on starch biosynthesis and granule structure in wheat. ### MATERIALS AND METHODS ### Plant Material and Cultivation The study was conducted at the Shihezi University Experimental Farm, Shihezi, China (44◦ 17′ N, 86◦ 03′ E) from October 2014 to June 2015. The soil at the site is classified as gray desert soil by Chinese scientists and a Calcaric Fluvisol according to the FAO. The 0–20 cm depth had the following characteristics: 63 mg kg−<sup>1</sup> available (mineral) N (potassium chloride extraction), 15 mg kg−<sup>1</sup> available P (Olsen), and 208 mg kg−<sup>1</sup> available K (ammonium acetate extraction). Seeds of the winter wheat cultivar "Xindong 20" were supplied by the Agriculture College of Shihezi University. On the day of sowing, 75 kg ha−<sup>1</sup> urea (46% N, Sinopec Group) was applied to the soil. The plots were drip irrigated at 10–12 d intervals three times before winter and six times after winter dormancy. Urea was applied via drip irrigation at elongation (45 kg ha−<sup>1</sup> ), heading (75 kg ha−<sup>1</sup> ), and flowering (120 kg ha−<sup>1</sup> ). The experiment used a randomized block design with three replications. Three P treatments were applied to the plots 160 d after sowing, when about 5% plants of the plants had turned green after dormancy. The P treatments were as follows: 0 kg P ha−<sup>1</sup> (control, abbreviated P0); 46 kg P ha−<sup>1</sup> (normal P, abbreviated NP); and 92 kg P ha−<sup>1</sup> (high P, abbreviated HP). The P fertilizer (triple superphosphate, 45% P) was applied in a 10-cm-deep band between each ridge. The plots were 2.4 × 3 m. Each plot was separated by a 50-cm-wide bare strip. ### Sampling Grain samples were collected from the middle region of the wheat spikes at 7, 14, 21, 28, and 35 days post anthesis (DPA). The samples were collected from each plot and then pooled to form one sample per treatment. Three subsamples were removed from each composite sample. These subsamples were frozen in liquid N for 5 min and then stored at −80◦C for RNA extraction. Another three subsamples were fixed in 4% paraformaldehyde (pH 7.2–7.6) and 0.1% DEPC for histochemical analysis and in situ hybridization. The remaining grains were dried at 70◦C to constant weight and weighed. Starch granules were isolated from grain (not oven-dried) collected at 35 DPA. ### Isolation of Starch Granules Starch granules were isolated using a modified version of the method used by Peng et al. (1999). The embryos were excised from mature wheat grains with a scalpel. The deembryonated grains were soaked overnight in deionized water at 4◦C. Subsequently, the grains were ground in a mortar and pestle with deionized water. The slurry was centrifuged (4,000 g, 10 min). The sediment was treated twice with 80% (w/v) CsCl. The starch was then washed three times with washing buffer (62.5 mmol L−<sup>1</sup> Tris-HCl, pH = 6.8; 10 mmol L−<sup>1</sup> EDTA; 4% w/v Abbreviations:* AGPase, adenosine diphosphate glucose pyrophosphorylase; AGP, gene encoding AGPase; AMY, gene encoding α-amylase; BAM, gene encoding β-amylase; CLSM, confocal laser scanning microscopy; DPA, days post anthesis; GBSS, granule-bound starch synthase; GBSS, gene encoding GBSS; HP, high phosphorus; ISO, gene encoding isoamylase; NP, normal phosphorus; P0, phosphorus application control; SBE, gene encoding starch branching enzyme; SEM, scanning electron microscopy; SS, gene encoding soluble starch synthase. SDS), three times with deionized water, and finally three times with acetone. The starch samples were then air dried. ### Starch Granule Morphology The starch granules were sprinkled onto double-sided conductive adhesive tape attached to aluminum stubs and then coated with gold-palladium (60:40) particles (20 nm particle size) using a sputter coater (Denton Vacuum-Moorestown, NJ, USA). The morphology of the starch granules was examined using a field emission scanning electron microscope (JEOL JFC-1600, Japan) at an accelerating voltage of 5–10 kV. ### Enzyme Assays Total amylase activity was measured as described by Liu (2011). Briefly, fresh, de-embryonated grains (1 g) were ground in 8 mL deionized water. The mixture was placed at room temperature for 15–20 min to extract total amylase. After centrifugation (3,000 rpm, 10 min), 1 mL of soluble starch (1%, w/v) was added to the supernatant (crude enzyme extract) and incubated at 40◦C for 5 min. Subsequently, 2 mL of 3, 5-dinitrosalicylic acid reagent (1% w/v 3,5-dinitrosalicylic acid, 30% w/v potassium sodium tartrate, and 0.4 mol L−<sup>1</sup> NaOH) was added to the mixture before heating in a boiling water bath. Absorbance was measured at 540 nm using a spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd.722G, China). A standard curve was prepared using malt sugar solutions with concentrations of 0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.0 mg mL−<sup>1</sup> . To measure α-amylase activity, β-amylase in the crude enzyme extract was inactivated by heating at 70◦C for 15 min. All of the other steps were the same as the assay of total amylase activity described above. Beta-amylase activity was calculated as the difference between total amylase and α-amylase activity. ### Treatment of Starch Granules with Proteolytic Enzyme The isolated starch granules (2 g) were suspended in 40 mL of proteolytic buffer [50 mmol L−<sup>1</sup> sodium acetate (pH 7.5), 1 mmol L −1 calcium chloride, and 0.02% (w/v) sodium azide]. Next, Protease Type XIV [196 units (g starch)−<sup>1</sup> , a mixture of various proteases, Sigma, Lot#051M1894V, USA, source: streptomyces griseus] was added to the suspension before incubating at 4◦C for 24 h on a shaking table. After centrifuging at 3,000 g for 20 min, Protease Type XIV-treated starch granules were washed with deionized water and ethanol and then air dried. ### Treatment of Starch Granules with Merbromin and 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) Protease (Type XIV)-treated starch granules were stained with a methanolic solution of merbromin and 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) according to Kim and Huber (2008). Merbromin is a non-reactive fluorescent dye that is absorbed onto the surface of the starch granules. Merbromin can be used to highlight external surface of the granules, including channels and cavities connected with the granule's exterior (Huber and BeMiller, 1997). The protein-specific dye CBQCA covalently reacts with primary amines of amino acids, peptides, and proteins under weak basic conditions. The dye, which is fluorescent only after reaction, can be used to highlight the protein network of radially oriented, channel-like structures within starch granules (Han et al., 2005). ### Confocal Laser Scanning Microscopy (CLSM) After staining with merbromin and CBQCA, the starch samples were transferred to glass slides and then photographed using a Zeiss LSM 510 CLSM system (Zeiss, Oberkoche, Germany). Excitation was achieved with an Argonlaser (488 nm) operating at 30% power. Emission was detected through an LP505 emission filter. ### Detection of Wheat Starch Content Grain starch content was determined according to the method of Zhao (2005), with three replications. Oven-dried grain was ground using a pulverizer (Shanghai Jiading Grain and Oil Instrument Co., Ltd. JFSD-70, China). Ten milligrams of the powder was transferred into centrifuge tubes and then blended with 100 µL of ethanol and 900 µL of 1 mol L−<sup>1</sup> NaOH. The mixture was heated in a boiling water bath for 10 min. After centrifugation (800 rpm, 15 min), 500 µl of the supernatant was diluted 200 times with distilled water, 1 mL of 1 mol L−<sup>1</sup> acetic acid and 1 mL iodine reagents (0.2% I2, 2% KI, w/v) were added, color development conditions were room temperature for 10 min, absorbance was measured at 620 nm with a spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd. 722G, China). A standard curve was prepared using soluble starch solutions at 0, 20, 40, 60, and 80% (w/v). ### Enzymatic Hydrolysis of the Starch Granules Alpha-amylase and amyloglucosidase digestion was conducted according to Tang et al. (2002). After isolation, the starch granules (25 mg) were suspended in 1 mL sodium acetate solution (0.1 mol L −1 , pH 4.8) containing 360U α-amylase (Sigma, A4551, USA) or 50U amyloglucosidase (Sigma, A7420, USA). The samples were incubated on a shaking incubator (200 rpm, 37◦C, 72 h). Next, 50 µL of 1 mol L−<sup>1</sup> HCl was added to the samples. The reaction was stopped by adjusting the pH to 7 using 1 mol L−<sup>1</sup> NaOH. The extent of starch degradation was determined by measuring the concentrations of reducing sugar produced by starch hydrolysis. Reducing sugar concentrations were determined using a modification of the method described by Bernfield (1951). After centrifuging at 1,500 g for 10 min, 0.1 mL of DNS reagent (0.63% w/v 3,5-dinitrosalicylic acid, 0.524 mol L <sup>−</sup><sup>1</sup> NaOH, 18.5% w/v NaKC4H4O6·4H2O, 0.5% w/v phenol, 0.5% w/v NaHSO3) was added to 0.1 mL of supernatant. The mixture was heated in a boiling water bath for 5 min. The absorbance was measured at 540 nm with a spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd.722G, China). A standard curve was prepared at using glucose at concentrations of 0, 100, 200, 300, 400, 500, and 600 µg mL−<sup>1</sup> . ### Detection of Relative Expression of Genes Involved in Starch Biosynthesis and Degradation ### Designation of Primers The primers of AGP1, AGP2, SS1, SS2, SS3, SS4, GBSS1, GBSS2, SBE1, SBE2A, SBE2B, ISO1, AMY1, AMY2, AMY3, AMY4, BAM1, BAM2, BAM3, BAM4, BAM5, BAM6, and BAM7 were designed using Primer Premier 5.0 software according to sequences published in the National Center for Biotechnology Information (NCBI). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reference control was wheat ACTIN gene. The specificity of the primers was tested and the PCR conditions were optimized using gradient PCR and agarose gel electrophoresis (Bio Rad, Power Pac 300, USA). The primer sequences are presented in Supplementary Table 1. ### RNA Extraction and cDNA Synthesis The RNA was extracted from de-embryonated wheat grain using RNAiso Plus (Takara, Cat#9108, Japan) and Fruit-mate (Takara, Cat#9192, Japan) kits according to the manufacturer's instructions. Total RNA quality was tested using agarose gel electrophoresis. First strand cDNA was synthesized using a reverse transcription kit (Tiangen, Cat#KR104-02, China). The cDNA quality was tested by amplifying the wheat ACTIN gene. ### Quantitative Real-Time PCR The rt-qPCR reaction solution was prepared with a SYBR Premix Ex Taq Kit (Takara, Cat#RR420A, Japan). The components are presented in Supplementary Table 2. The amplifications of the individual cDNA sequences were detected using real time qPCR (Roche LightCycler 480 II, USA) with three replications. A mathematical model was used to determine the relative expression of target gene compared with the reference gene. The relative quantification was calculated with the following formulae: The wheat ACTIN gene was used as the reference. The gene is the favored reference for studying wheat genes because it is highly conserved in cell integrity, motility, and structure. ### In Situ Localization of AMY4, BAM1, and BAM5 Transcripts ### Synthesis of the Probes The AMY4, BAM1, and BAM5 gene fragments were amplified using PCR. The fragments were collected using an EasyPure Quick Gel Extraction Kit (Transgen, Code #EG101-01, China). The fragments were linked with pEASY-T3 cloning vectors, and then the vectors were transformed to pEASY-T1 competent cells using a pEASY-T3 cloning kit (Transgen, Cat#CT301- 1, China). After screening and culturing, the plasmids were isolated using a TIANprep Mini Plasmid Kit (Tiangen, Cat #DP103-02, China) and sequenced by Sangon Biotech (Shanghai) Co., Ltd. Based on the sequencing results, the plasmids were digested with a restriction enzyme, either Nco I (Takara, Code#1160A, Japan, 10U µl −1 , source: Escherichia coli carrying the plasmid encoding Nco I gene) or Pst I (Takara, Code#1073A, Japan, 15U µl −1 , source: Escherichia coli ED8654 carrying the plasmid encoding Pst I gene). The linearized plasmids were used to synthesize antisense and sense probes via in vitro transcription. This was performed using a Dig RNA Labeling Kit (Roche, REF11175025910, USA). The antisense probe sequence of AMY4 was as follows: 5 ′ -UUGGUUUCCGAUGGUGUUGUCCAAGAACAG GCAGCUCGCAAUGGCGGGAUCAUUAAGAACGGGAGA GAAAUCCUAUUGCAGGCUUUUAAUUGGGAAUCCCA UAAACACAAUUGGUGGAGUAAUUUAGAGGGCAGAGU UGCCGACAUUGCUA-3′ . The antisense probe sequence of BAM1 was as follows: 5 ′ -ACUCAGGAAUGCAAGGCCUCAUGGCAUCAACAAG AGCGGCCCUCCUGAGCACAAGCUGUUUGGAUU CACCUACCUCCGGCUGUCGAAUCAGUUGGUGGAG GGACAAAACUAUGUCAAUUUCAAGACCUUUGUUGACA GAAUGCAUGCCAACCUGCCUCAUGACCCAU-3′ . The antisense probe sequence of BAM5 was as follows: 5 ′ -UGAACCGGAACCUGUUCGACGGCGACAACU GGCGACGGUUCGUCGCGUUCGUGAAGACCAUG GCCGACGGCGGCGCGAGGACGGCGCUGCCCAGGU GCGACACUGGGCACUCGGAUCUGUACGUGGGGUUC GUUGA-3′ . ### Paraffin Sectioning The method for making a paraffin section was modified from Ausubel et al. (1995). Wheat grains were crosscut and fixed in 4% paraformaldehyde (pH 7.2–7.6) and 0.1% DEPC under slight vacuum for 4 h at room temperature. The grains were dehydrated in a graded alcohol series (30–100%), and then cleared three times in solutions of alcohol and chloroform with ratios of 3:1, 1:1, and 1:3, each time for 3 h. Finally, the grains were cleared in absolute chloroform for 3 d. The cleared grains were infiltrated with a graded mixture of chloroform and paraffin wax at different temperatures (3:1, 40◦C; 1:1, 40◦C; 1:3, 45◦C), each time for 4 h. Then, the grains were infiltrated in paraffin wax at 55◦C either for 3 d (grain collected of 21 DPA and before) or for 5 d (grain collected at 28 and 35 DPA). The infiltrated grains were embedded in paraffin wax and then sectioned into 10–25µm thicknesses on a microtome (Kedee, 1508A, China). The exposed surfaces of grain collected at 28 and 35 DPA were soaked in DEPC-water for several hours before being sectioning. The sections were then affixed to adhesive microscope slides (Citoglas, REF188105W, China). ### In Situ Hybridization The paraffin sections were dewaxed and rehydrated. Then, in situ hybridization was performed with an Enhanced Sensitivity ISH Detection Kit I, POD (Boster, MK1030, China). The paraffin sections were observed and photographed using a stereo microscope (Zeiss Discovery V20, Germany). ### Staining of Grain Median Transverse Sections with I2-KI Paraffin sections containing wheat grain were stained with I2-KI (0.1%) for 5 min after dewaxing and rehydration. The sections were washed with deionized water, and then observed and photographed using a stereo microscope (Zeiss Discovery V20, Germany). ### Statistical Analysis and Image Processing The data was analyzed by one-way ANOVA using Microsoft Excel and SPSS 13.0 software. Significance comparisons were made by Duncan's multiple range test at P < 0.05. Image processing was performed using Adobe Photoshop CS6. ### RESULTS ### Starch Content and Starch Granule Morphology Grain weight increased across time and was significantly enhanced by P application (Figure 1). Similarly, the total starch content in the grain was very low during the early grain-filling stage and then increased with grain development. Beginning at 14 DPA, total starch contents were significantly greater in HP and NP than in P0 (note the difference between NP and P0 was not significant on 21 DPA). The grain also matured earlier in HP and NP than in P0 (Supplementary Figure 1). The morphological characteristics of the starch granules was observed using SEM (Figures 2A–C). The A-type granules were disk-shaped with diameters >10µm. The B-type granules were spherical with diameters <10µm. The "pinholes" along the equatorial grooves of the granules and on their flat surfaces were more obvious in NP than in HP and P0. To study the effects of P fertilizer on the micro-structure of starch granules, protease (Type XIV) -treated granules were stained with methanolic merbromin and CBQCA and then visualized by CLSM. The results showed that P fertilizer caused substantial changes in the histochemical patterns of the starch granules. Fluorescence was clearly visible and strong in large areas of many starch granules in NP (Figures 2D–F). In contrast, the P0 samples exhibited only faint fluorescence at the equatorial regions. The fluorescence of starch granules in HP was intermediate between NP and P0. The CBQCA staining (Figures 2G–I) patterns were similar those of membromin. These results suggested that P application influenced, presumably enhanced the pits and channels within starch granules. ### Reducing Sugars from Starch Granules after Exogenous Enzymatic Hydrolysis Drought-induced microstructural changes to starch granules may facilitate the transfer of chemicals (water, enzymes, and acid) into the matrix of the starch granule and accelerate hydrolysis (Li et al., 2015). To ascertain whether the effects of P application were similar to those of drought, we measured the amounts of reducing sugars released from granules after hydrolysis for 72 h with amyloglucosidase and α-amylase. Reducing sugar concentrations after digestion were greater in HP and NP than in P0 (Figure 3, note the difference was not significant between HP and P0 in the amyloglucosidase treated samples). Together with the SEM and CLSM images, this result suggested that P fertilizer enhanced the pits and channels in starch granules and increased the starch surface area available for hydrolysis reactions. ### Patterns of α-Amylase and β-Amylase Activities during Grain Filling The α- and β-amylase activities in the grain varied depending on sampling time and P treatment (Figure 4). The β-amylase activity was much higher than the α-amylase activity. The α-amylase activity under three treatments was gradually decreased from 7 FIGURE 2 \| Starch granules isolated from mature wheat seeds (35 days post anthesis) and observed using SEM (×500 magnification) and CLSM (×400 magnification). Starch granules were isolated from the wheat grain in the 0 kg P ha−<sup>1</sup> (P0) treatment (A,D,G), 46 kg P ha−<sup>1</sup> (NP) treatment (B,E,H), and 92 kg P ha−<sup>1</sup> (HP) treatment (C,F,I). (A–C), starch granules observed using SEM; (D–F) starch granules stained with merbromin and then observed using CLSM; (G–I) starch granules stained with CBQCA and then observed using CLSM. The "pinholes" along the equatorial grooves and on flat surfaces of the granules were visualized with box. The magnified insets (×2,000) (A–C) were the starch in red box. Arrows indicate short channels and/or cavities (connected to the exterior by channels) (E,F) and radially oriented, channel-like, protein networks (G–I) within the granules. to 21 DPA and then remained steady. The α-amylase activity was significantly greater in NP than in HP at 7, 21, 28, and 35 DPA. The P0 treatment had the lowest α-amylase activity among the treatments (except for 28 DPA). The β-amylase activity in all three P treatments gradually increased from 7 to 28 DPA and then declined. The β-amylase activity was significantly greater in HP and NP than in P0 at 21 and 28 DPA; however there was no difference between HP and P0 at 35 DPA. ### Patterns of Expression of Genes Involved in Starch Synthesis and Degradation during Grain Filling The relative expressions of genes involved in starch synthesis and degradation are shown in Figures 5, 6. The expression patterns of both AGP1 and AGP2 were similar across time in all three P treatments. The NP treatment had the highest AGP1 and AGP2 expression at 7 and 14 DPA. The expression pattern of GBSS1 was different that of GBSS2. The GBSS1 transcripts were most abundant at 21 and 28 DPA, whereas the GBSS2 transcripts were most abundant at 7 and 14 DPA. The NP treatment had the highest GBSS1 expression (at 21 DPA) and the highest GBSS2 expression (at 7 DPA). In NP, the SS1, SS2, and SS3 transcripts were greatest at 7 and 14 DPA and then decreased across time. In contrast, the SS4 transcripts remained steady between 7 and 21 DPA and then increased significantly at 28 DPA. In HP, the SS1, SS2, and SS3 transcripts were gradually increased from 7 to 14 DPA, whereas SS4 showed little expression on any sample date. Among the genes encoding starch branching enzyme (SBE1, SBE2A, and SBE2B), SBE1 transcripts were most abundant. In NP, SBE1 transcript abundance was greatest at 21 DPA, whereas the transcript abundances of SBE2A and SBE2B were both greatest at 7 DPA and then decreased. In HP, the transcripts of SBE1, SBE2A, and SBE2B were gradually increased from 7 to 14 DPA then decreased during the remaining time. The NP and HP treatments generally upregulated the ISO1 transcripts, especially at 14 DPA. The NP treatment upregulated the AMY1 transcripts compared with P0, with peak expression at 21 DPA. In contrast, HP downregulated AMY1. The transcript levels of AMY2 were significantly greater in NP than in P0 and HP. There was no significant different in AMY2 expression between P0 and HP between 7 and 35 DPA. The relative expression of AMY3 in both P0 and NP was high and significantly greater than that in HP between 7 and 35 DPA. The relative expression of AMY4 increased suddenly at 28 DPA in all three P treatments. The AMY4 expression was greater in NP than in P0 and HP. The seven BAM genes were differentially expressed among the P treatments. The BAM transcript levels were greater in NP than in P0 and HP. In NP and HP, BAM1 was mostly highly expressed at 28 DPA, whereas BAM2 was most highly expressed at 7 DPA. Of the expression patterns of BAM3 and BAM5 were almost identical both in NP and in HP. However, BAM3 and BAM5 genes were weakly expressed in P0. The transcript pattern of BAM4 significantly varied among the P treatments. The BAM4 expression at 7 and 14 DPA was less than that on the other sample dates. The transcript patterns of BAM6 and BAM7 were similar. The NP treatment upregulated both genes, with relative gene expression reaching a maximum at 21 DPA. The HP treatment downregulated BAM6 but upregulated BAM7 compared with P0. These results indicated that 12 genes involved in starch synthesis and 11 genes involved in starch degradation were expressed in the developing wheat grains. Furthermore, the P treatments significantly influenced the expression patterns of these genes. Compared with P0 and HP, NP upregulated genes encoding starch synthesis enzymes (especially during early to mid-grain filling) and starch degradation enzymes (especially during mid- and late-grain filling). ### Spatial Profiling of Transcripts of AMY4, BAM1, and BAM5 during Grain Filling As mentioned previously, β-amylase activity was much greater than α-amylase activity, and the transcription levels of BAM1 and BAM5 were the highest among the seven BAM genes. In addition, the relative expression of AMY4 increased sharply to high levels during late grain filling. For these reasons, AMY4, BAM1, and BAM5 mRNA were localized using in situ hybridization (anti sense: Figure 7, Supplementary Figures 2, 3; sense control: Supplementary Figures 4–6). Starch accumulation in wheat caryopses (Figures 7D,H,L; Supplementary Figures 3D,H,L) indicated that the cavity in the ventral groove of caryopses was an intrinsic characteristic of wheat grains. AMY4, BAM1, and BAM5 transcripts were detectable in both the pericarp and early endosperm at 7 DPA in all three P treatments (Supplementary Figure 2). In P0, AMY4, BAM1, and BAM5 transcripts were detected in the entire endosperm from 7 to 35 DPA (Figures 7A–C, Supplementary Figures 3A–C). In NP and HP, AMY4, BAM1, and BAM5 transcripts had accumulated at the endosperm border at 28 DPA (Figures 7E–G,I–K) and at 35 DPA (Supplementary Figures 3E–G,I–K). However, the relative expression of AMY4, BAM1, and BAM5 at the edge of the endosperm was greater in NP (Figures 7E–G, Supplementary Figures 3E–G) than in HP (Figures 7I–K, Supplementary Figures 3I–K). This result showed that P fertilizer increased the transcript abundance of amylase genes at the edge of the endosperm. This phenomenon was more pronounced in NP than in HP. ### DISCUSSION Wheat endosperm is the main tissue for biosynthesis and accumulation of starch. The A-type and B-type starch granules in mature wheat endosperm display bimodal distribution (Evers, 1971). Based on anatomical studies, Parker (1985) reported that A-type starch granules in wheat were initiated between ∼4 and 14 DPA. The B-type starch granules appeared from about 14 DPA until grain maturity. Previous research in our laboratory showed that P fertilizer significantly influenced the ratio of A/B starch granules and the average diameter of the granules (Li et al., 2013). However, the SEM images in this study showed that P fertilizer did not cause significant changes in the shape of the starch granules. Overall, these results indicate that P application may affect the timing and development of A- and B-type starch granules in wheat. Analysis of the expression patterns of genes involved in starch synthesis is important to understand the mechanism of starch biosynthesis. A previous study showed that AGPase is less sensitive to 3-PGA and inorganic P in cereal endosperm than in other tissues (Gómez-Casati and Iglesias, 2002). This suggests AGPase activity may be controlled at the transcriptional level in endosperm. McCue et al. (2002) found that GBSS I may control starch synthesis at the transcriptional and post-transcriptional level. Wang et al. (2014) studied the relationships among starch accumulation, the activities of key enzymes, and gene expression in wheat endosperm. The results indicated the amylose, amylopectin, and total starch accumulation rate were significantly and positively correlated with the activities of SBE, SSS, and GBSS. The SBE, SSS, and DBE may control starch synthesis at the transcriptional level, whereas GBSS1 may control starch synthesis at the post transcriptional level. Phosphorus increases photosynthetic rates and promotes post anthesis dry matter accumulation (Zhu et al., 2012). Both of these factors play a vital role in starch biosynthesis and accumulation. In the present study, P application increased the expression of genes involved in starch synthesis, and these increases in gene expression coincided with greater starch content. This indicated that P application promoted starch biosynthesis not only by increasing photosynthetic rates to produce more substrate, but perhaps also by controlling starch synthesis at the transcriptional level. However, excess P application may also enhance respiration, which could increase sugar and energy loss (Shen, 2001). It is well-known that P application can cause earlier seed maturity, and we observed that the grain was darker (or more yellow) in HP than in NP and P0 at 35 DPA (Supplementary Figure 1). This indicates that the grain filling stage in HP was shorter than that in the other two treatments. This perhaps is one reason why HP had less starch accumulation than NP. Whan et al. (2014) studied α-amylase levels in wheat grain and suggested the endosperm-specific over-expression of AMY3 resulted in an increase in total α-amylase activity in harvested wheat grain. However, increased α-amylase activity did not significantly influence starch content or composition. As mentioned previously, seed maturity can be promoted by P application (Supplementary Figure 1). In certain varieties of wheat, triticale, and barley, amylase activity always increases with grain maturity (Lindblom et al., 1989; Mares and Oettler, 1991; Radchuk et al., 2009). Thus, it is possible that increased amylase activity in this study resulted from early maturity induced by P application. On the other hand, the increased expression of amylase genes in NP and enhanced amylase activity suggested that P may also control starch degradation at the transcriptional level. Whan et al. (2014) observed that enhanced amylase activity did not reduce starch content. Therefore, there is no contradiction between high amylase activity and high starch content. Increases in amylase activity may cause variation in the channel structures of starch granules. The channels and pores within starch granules are intrinsic characteristics of wheat. Using CLSM, Kim and Huber (2008) observed that the channel types are different in A- and Btype granules of wheat starch wheat. External factors [e.g., high temperature (Li et al., 2017) and drought stress (Li et al., 2015)] can enhance the number and size of the channels. In this study, "pinholes" and pits on flat surfaces along the equatorial groove of starch granules were more obvious in NP than in HP and P0. The CLSM images and the enzymatic digestion results also confirm such micro-structural changes. We speculate that the increases in channel size and number enlarge the granule surface area available for hydrolysis reactions, resulting ultimately in the release of more glucose units. Earlier investigations in our laboratory revealed that pit micro-structures affected such starch characteristics as pasting properties, swelling power, solubility capacity, and enzymatic hydrolysis (Li et al., 2013, 2015). On the other hand, P-induced changes in the physicochemical properties of starch are complex, and other factors also have significant influence (e.g., the ratio of A- and B-type granules, as well as their volume, internal structure, and surface area; Hayfa et al., 2009). Fannon et al. (2004) postulated that in the endosperm of sorghum and maize, amyloplasts contain microtubules which radiate outward from the initiation point of starch granule growth (i.e., granule hilum) to the plastid periphery. The authors speculated that the granule develops around the radially-oriented microtubules, which become channels terminating at the outer surface of starch granules. Therefore, the channels within starch granules are the remnants of amyloplast microtubules. Benmoussa et al. (2010) observed that the protein constituents of channels in maize starch included actin-like and tubulin-like structural proteins, a membrane protein (adenylate translocator, Bt1), and the enzymes involved in starch biosynthesis. Further, Benmoussa et al. (2010) hypothesized that microtubules may possess at least two purposes in amyloplasts and starch granules: (1) they may facilitate starch polymer and granule biosynthesis; and (2) they may function to provide variation in the process of granule degradation during seed germination (Fannon et al., 1992a,b). was 20µm. We are not aware of any previous studies which have examined the biological importance of pits and channels in wheat starch biosynthesis. Based on previous findings in maize, we hypothesize that P fertilizer promotes the development of amyloplasts and influences the structure of amyloplast microtubules (Benmoussa et al., 2010). The microtubules may provide greater surface area for transporting starch-synthesizing enzymes and substrates needed for starch synthesis into the amyloplast. The hypothesis is supported by the observation that NP significantly increased wheat grain starch content as well as the expression of genes involved in starch synthesis. Furthermore, the observation that starch content was greater in NP than in HP suggests that NP optimized the exchange of substances for starch biosynthesis. During late grain filling, increasing α-amylase and β-amylase activities may act on the channel ends to form pits on the granule surfaces. A previous study in our laboratory showed that endogenous hydrolysis (seed germination) was increased by P-induced increases in the number of pits and channels in wheat starch granules (Zu et al., 2017). Our observations appear to confirm the proposal by Benmoussa et al. (2010) that channels may influence granule degradation during seed germination. Further study is necessary to test this hypothesis. Such studies may provide significant information about the relationship between P application and starch biosynthesis. Wheat endosperm cells are differentiated from the meristematic region at the periphery of the endosperm. Therefore, the youngest cells are found at the outer edge of the endosperm and the oldest at the center (Bradbury et al., 1956). During wheat grain development, starchy endosperm initiates a cell death program (Young and Gallie, 2000). In this study, P application increased (i) the expression of amylase genes; (ii) amylase activity; and (iii) AMY4, BAM1, and BAM5 transcript abundance at the periphery of the endosperm at 28 DPA. This indicates that during late grain filling in NP, the meristematic region at the periphery of the endosperm still maintained metabolic activities to support the relatively abundant transcripts of genes involved in starch synthesis and degradation. This explains the increased starch content in NP. ### CONCLUSIONS The results of this study show that P fertilizer significantly altered microstructures in the starch granules. This is important because the channels may provide greater surface area for the transport of starch-synthesizing enzymes and substrates needed for starch synthesis. The study also indicated that P fertilizer significantly affected starch accumulation by influencing the expression of genes related to starch biosynthesis and degradation. Further study is necessary to understand the mechanism by which P influences starch morphology and biosynthesis. Such information may provide information helpful for increasing wheat yield and starch quality. The latter could have important implications for the food industry. ### AUTHOR CONTRIBUTIONS RZ: Substantial contributions to the acquisition, analysis, and interpretation of data for the work; Drafting the work and revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. KF, CAL: Substantial contributions to the acquisition, analysis, and interpretation of data for the work; Revising the work critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CEL, CYL: Substantial contributions to the conception and design of the work; Drafting the work and revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ### ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (31360334, 31160256, 31360292, and 31560389), New Cultivar Breeding and Germplasm Enhancement of Wheat (2016AC027), Young Innovator ### REFERENCES Cultivating Project of Shihezi University (CXRC201703), and Specific Project for Breeding of Shihezi University (YZZX201702). ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017. 02252/full#supplementary-material Supplementary Table 1 \| Characteristics of primers used to measure differential gene expression using Quantitative PCR. Supplementary Table 2 \| Components and volume of real time quantitative PCR reaction solution. Supplementary Figure 1 \| Fresh grains at 28 DPA (A–C) and 35 DPA (D–F) under P0 (A,D), NP (B,E), and HP (C,F) conditions. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . Supplementary Figure 2 \| In situ localization of AMY4, BAM1, and BAM5 transcripts in wheat caryopses at 7 DPA (×30). Hybridization sites of AMY4 (A,D,G), BAM1 (B,E,H), and BAM5 (C,F,I) transcripts were visualized as reddish-brown signals in median transverse sections of wheat grains under the P0 (A–C), NP (D–F), and HP (G–I) conditions, respectively. es, endosperm; np, nucellar projection; p, pericarp. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . The thickness of these sections was 20µm. Supplementary Figure 3 \| In situ localization of AMY4, BAM1, and BAM5 transcripts and starch accumulation in wheat caryopses at 35 DPA (×25, the magnified insets ×150). Hybridization sites of AMY4 (A,E,I), BAM1 (B,F,J), and BAM5 (C,G,K) transcripts were visualized as reddish-brown signals in median transverse sections of wheat grains under the P0 (A–C), NP (E–G), and HP (I–K) conditions, respectively. Starch granules were stained with I2-KI in median transverse sections of wheat grains under the P0 (D), NP (H), and HP (L) conditions, respectively. al, Aleurone; es, endosperm; np, nucellar projection. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . The thickness of these sections was 20µm. Supplementary Figure 4 \| Sense control of AMY4, BAM1, and BAM5 transcripts in wheat caryopses at 7 DPA (×25). Sense control of AMY4 (A,D,G), BAM1 (B,E,H), and BAM5 (C,F,I) transcripts in median transverse sections of wheat grains under the P0 (A–C), NP (D–F), and HP (G–I) conditions, respectively. es, endosperm; np, nucellar projection; p, pericarp. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . The thickness of these sections was 20µm. Supplementary Figure 5 \| Sense control of AMY4, BAM1, and BAM5 transcripts in wheat caryopses at 28 DPA (×25, the magnified insets ×150). Sense control of AMY4 (A,D,G), BAM1 (B,E,H), and BAM5 (C,F,I) transcripts in median transverse sections of wheat grains under the P0 (A–C), NP (D–F), and HP (G–I) conditions, respectively. al, Aleurone; es, endosperm; np, nucellar projection. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . The thickness of these sections was 20µm. Supplementary Figure 6 \| Sense control of AMY4, BAM1, and BAM5 transcripts in wheat caryopses at 35 DPA (×25, the magnified insets ×150). Sense control of AMY4 (A,D,G), BAM1 (B,E,H), and BAM5 (C,F,I) transcripts in median transverse sections of wheat grains under the P0 (A–C), NP (D–F), and HP (G–I) conditions, respectively. al, Aleurone; es, endosperm; np, nucellar projection. P0: 0 kg P ha−<sup>1</sup> ; NP: 46 kg P ha−<sup>1</sup> ; HP: 92 kg P ha−<sup>1</sup> . The thickness of these sections was 20µm. endosperm amyloplasts. J. Cereal Sci. 52, 22–29. doi: 10.1016/j.jcs.2010. 02.013 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Zhang, Li, Fu, Li and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Common Bean Fe Biofortification Using Model Species' Lessons Raul A. Sperotto<sup>1</sup> \* and Felipe K. Ricachenevsky 2, 3 \* <sup>1</sup> Biological Sciences and Health Center, Graduate Program in Biotechnology, University of Taquari Valley - UNIVATES, Lajeado, Brazil, <sup>2</sup> Graduate Program in Agrobiology, Biology Department, Federal University of Santa Maria, Santa Maria, Brazil, <sup>3</sup> Graduate Program in Cell and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Keywords: anti-nutrient, bean, biofortification, iron, model species, Phaseolus vulgaris, transgenic strategies Common bean (Phaseolus vulgaris L.) is the most widely grown grain legume for direct human consumption and is highly preferred in many parts of Africa and Latin America, as well as in southern Europe (Broughton et al., 2003; Blair and Izquierdo, 2012). It is an important source of nutrients for more than 300 million people, representing 65% of total protein consumed, 32% of energy, and a major source of micronutrients e.g., iron (Fe), zinc, thiamin, and folic acid (Welch et al., 2000; Broughton et al., 2003; Blair et al., 2010a; Petry et al., 2015). It is known as the "poor men's meat," due to its high protein, minerals, and vitamins content (Blair, 2013). Fe is an essential micronutrient for almost all living organisms (Bashir et al., 2013), and Fe deficiency is the most common micronutrient deficiency worldwide, disproportionately affecting the poorest and most vulnerable populations in resource-limited settings, leading to Fe deficiency anemia (IDA; Stevens et al., 2013; Finkelstein et al., 2017). IDA is difficult to address through Fe supplementation or processed foods; therefore, several attempts are being made to enhance Fe accumulation into staples such as rice, maize, wheat, and legumes (Blair and Izquierdo, 2012) using biofortification, which is the process of breeding or genetic engineering to improve nutrient content in a crop (Blair, 2013). Biofortification is considered a sustainable and cost effective strategy to address malnutrition in developing countries because it targets staple foods that are consumed daily (Dwivedi et al., 2012). ### Edited by: Sebastien Thomine, Centre National de la Recherche Scientifique (CNRS), France #### Reviewed by: Louis Grillet, Academia Sinica, Taiwan \Correspondence: Raul A. Sperotto [email protected] Felipe K. Ricachenevsky [email protected]* #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 23 October 2017 Accepted: 12 December 2017 Published: 22 December 2017 #### Citation: Sperotto RA and Ricachenevsky FK (2017) Common Bean Fe Biofortification Using Model Species' Lessons. Front. Plant Sci. 8:2187. doi: 10.3389/fpls.2017.02187 Nutritional quality in common beans has been found to be higher than in cereals, with large amounts of minerals and vitamins accumulated in the seeds (Broughton et al., 2003). Common bean is estimated to have 4–10 times the amount of Fe, and 2–3 times the amount of Zn compared to rice (Pfeiffer and McClafferty, 2007). Also, these minerals and vitamins are generally retained after harvest and processing, while in polished cereal grains the Fe-rich tissues (embryo and aleurone layer) are lost (Wang et al., 2003). Although the average Fe concentration in beans is high, many people still suffer from IDA due to an insufficient level of bioavailable Fe in a monotonous cereal/bean-based diet without meat (Bouis, 2007). For Fe biofortification purposes, the use of common bean is advantageous because the baseline grain Fe content is high at 55 ppm and variability for the trait is great (Petry et al., 2015), ranging up to 110 ppm, allowing initial biofortification attempts to start from already high Fe levels (Blair et al., 2012; Blair, 2013). Another advantage of using common beans in biofortification programs is that seeds are consumed whole after boiling. Therefore, all major components of the common bean seed could be targets of biofortification: seed coat, cotyledons, and embryo (Blair et al., 2013). The target Fe level of HarvestPlus, an international research program supporting the research and development of biofortified crops, is 94 ppm in whole bean seeds (Blair and Izquierdo, 2012; Petry et al., 2015). According to Vasconcelos et al. (2017), in order to achieve 30% of the estimated average daily dietary requirement for Fe on a dry weight (DW) basis, the recommended Fe levels in whole beans should be 107 ppm. The target level was quickly reached, and in some countries plant breeders have already developed and released new P. vulgaris bean varieties with Fe concentrations of about 100 ppm (Petry et al., 2015). These varieties show good micronutrient retention after processing, and equal or increased agronomic yield (Bouis and Welch, 2010). However, successful 328 bean Fe biofortification might be constrained due to the reported low Fe bioavailability (Ariza-Nieto et al., 2007) associated with high concentrations of Fe absorption inhibitors, also called antinutrients, such as polyphenols and phytate (Beninger et al., 2005; Petry et al., 2014). Here we propose multiple, complementary approaches to increase Fe concentration and bioavailability in common bean, based on the current knowledge on model species. These approaches are summarized in Figure 1. ### DECREASING ANTI-NUTRIENT CONCENTRATION AND CO-LOCALIZATION WITH FE IN SEEDS Short-term human isotope studies indicate that phytate is the major Fe absorption inhibitor in beans, with polyphenols playing a minor role (Petry et al., 2012, 2014). Phytate increases with the Fe concentration in beans, and both are mainly found in the cotyledons. It should be possible to simultaneously breed for high Fe and low phytate since most phytate-related QTLs are independent of Fe concentration QTLs (Blair et al., 2012, 2013). Two main strategies for phytate reduction in seeds have been attempted: disruption of its biosynthetic pathway with knockout/knockdown of inositol pentakisphosphate 2-kinase (IPK1) in Arabidopsis and rice showing decreased phytate in seeds and normal yield (Stevenson-Paulik et al., 2005; Ali et al., 2013), but with possible defects in Pi homeostasis (Kuo et al., 2014); and mutations on phytate vacuolar transporters expressed in seeds, which reduced phytate concentration in other species (Shi et al., 2007; Nagy et al., 2009; Xu et al., 2009). In common bean, disruption of the orthologous transporter PvMRP6 resulted in 90% less phytate in seeds and normal complementary, non-excludent approaches for bean biofortification. (A) Schematic representation of common bean seed and its main parts: seed coat, embryo and cotyledons. In cotyledons, iron (Fe) is shown with phytate (PA), whereas in the seed coat (detail), Fe is shown with polyphenols (PP). Each one act as an Fe absorption inhibitor in the human gut, with PA being likely a stronger anti-nutrient than PP. (1) Strategy aiming at increasing Fe concentration in the cotyledons to overcome PA anti-nutrient properties. (2) Strategy aiming at decreasing PA concentration in the cotyledons, making Fe in this tissue more bioavailable. (3) Strategy aiming at increasing Fe concentration in the seed coat to overcome PP anti-nutrient properties. (4) Strategy aiming at decreasing PP concentration in the seed coat, making Fe in this tissue more bioavailable. (B) Proposed candidate genes for genetic engineering in common bean, based on previous studies in model species. These genes are orthologous to genes found in Arabidopsis thaliana based on BLAST searches, except for Phvul.001G012200, which is the best hit using a soybean (Glycine max) IPK gene as query (Yuan et al., 2012). For each candidate gene, the type of manipulation is indicated. agronomic performance (Panzeri et al., 2011; Campion et al., 2013). However, seeds were hard to cook and induced digestive problems in human subjects (Petry et al., 2016). Thus, further research is necessary to improve Fe bioavailability by decreasing phytate while maintaining agronomic performance and consumer preferences. Biofortification in beans can target all seed tissues: the thick seed coat, two large cotyledons and a well-developed embryo (Blair et al., 2013), which comprise 7–10, 85, and 2–3% of seed weight, respectively (Ariza-Nieto et al., 2007). Remarkably, segregating populations derived from crosses between wild and cultivated parents showed that QTLs for Fe accumulation in each tissue segregate separately, and the Fe range and maximum amount observed in seed coat is higher than in cotyledons (Blair et al., 2013). Seed coat can contribute with as much as 26% of the total seed Fe, and polyphenols, not phytate, are the main anti-nutrients in the tissue (Ariza-Nieto et al., 2007). Thus, exploring seed coat biofortification is promising, as little is known about which specific polyphenol molecules reduce Fe bioavailability and how reduction in their concentration might affect plant and seed physiology (Petry et al., 2015). ### FURTHER INCREASING FE ACCUMULATION IN BEANS Genetic engineering beans to accumulate higher Fe concentrations in seeds can benefit from work on model plants. Vacuolar Iron Transporter (VIT) family members are likely candidates, since they are involved in seed Fe localization and/or concentration in Arabidopsis and rice (Kim et al., 2006; Zhang et al., 2012). AtVIT1 localizes Fe to the provasculature, and changes in provasculature density have been proposed as a means to increase Fe content in seeds (Roschzttardtz et al., 2017). In rice, OsVIT1 and OsVIT2 are involved in flag leaf Fe pool regulation, and might also have a role in seed Fe localization (Zhang et al., 2012). Recent work showed that endosperm-specific overexpression of TaVIT2 increased Fe concentration in wheat endosperm (Connorton et al., 2017), indicating that VIT genes can increase tissue Fe sink strength. In rice, overexpression of NICOTIANAMINE SYNTHASE (NAS) genes was shown to substantially increase Fe concentration in the endosperm, presumably increasing Fe translocation through the phloem (Johnson et al., 2011). In addition, OsNAS1 over-expression in rice plants enhance human Fe bioavailability from the high nicotianamine (NA) grains (Zheng et al., 2010). NA role in Fe long-distance transport is likely to be conserved in land plants (Schuler and Bauer, 2011), and therefore a similar approach could be applied to common bean. Wirth et al. (2009) overexpressed bean Ferritin, Arabidopsis Nicotianamine synthase, and Aspergillus fumigatus Phytase genes in rice plants, and detected 6.3 fold increase in Fe concentration on the polished seeds. Aluru et al. (2011) used a lpa maize mutant to overexpress soybean Ferritin gene, and found 2.7-fold increase in seed Fe concentration. Similar approaches could be certainly used in common bean plants in order to concomitantly decrease phytate levels and increase Fe accumulation and availability. Another approach would be to explore genes that regulate Fe concentration. Over-expression of AtbHLH039 results in constitutive Fe deficiency response and increased Fe levels in leaves and seeds (Naranjo-Arcos et al., 2017). Interestingly, the bean genome has only one gene similar to all four subgroup Ib from Arabidopsis, which are known to be involved in Fe deficiency response (Brumbarova et al., 2015). Work in Arabidopsis and rice has shown that the negative regulators of Fe deficiency response BRUTUS/HRZ-like proteins could lead to increased Fe concentration in seeds of knockout/knockdown plants (Kobayashi et al., 2013; Hindt et al., 2017). Three BRUTUS/HRZ-like genes are found in the bean genome. Although promising, manipulation of regulatory proteins should be performed with caution, since plants might display undesired phenotypic changes besides increased Fe in seeds. Common bean genetic transformation protocols are lengthy and of low reproducibility, while in vitro plant regeneration is especially difficult (Veltcheva et al., 2005; Rech et al., 2008). The Agrobacterium rhizogenes system allow for bean root transformation and could be used for characterization and selection of candidate genes for stable transformation (Estrada-Navarrete et al., 2007). Another solution is CRISPR-Cas9 mediated genome editing, which does not necessarily require transformation (Malnoy et al., 2016; Wolt et al., 2016) and could circumvent the problem in the near future. However, using this method, it would be easier to knockout a specific gene than overexpress it. ### EXPLORING BEAN NATURAL VARIATION AND WILD RELATIVES The wide genetic Fe variability of beans has enabled plant breeders to develop varieties with twice Fe compared to normal beans (Blair et al., 2010b). Common bean is native to Latin America, and is one of the five cultivated species of the Phaseolus genus. It has two main genetic pools: Andean (large seeds) and Mesoamerican (small seeds). Andean and intergene-pool hybrids have higher Fe concentrations compared to Measoamerican ones, although the range of variation is similar (Blair, 2013). Large germplasm collection screenings for high Fe genotypes conducted in local and wild varieties of P. vulgaris have reported up to 110 ppm Fe. However, early analyses on closely related species such as P. coccineus and P. dumosus have found up to 127 ppm Fe, indicating that wild relatives might be useful (Blair et al., 2013). Even considering that high Fe wild genetic material showed poor agronomical performance (and introgression might not be straightforward in interspecific crosses), further screening of wild genotypes is promising. Moreover, wild beans accumulate more Fe in seed coats and less in cotyledons compared to domesticated genotypes, indicating that they can contribute differently for tissue-specific biofortification (Blair et al., 2013). QTL studies show that multiple genes regulate seed Fe levels (Blair and Izquierdo, 2012; Blair et al., 2013). Interestingly, Fe concentration inheritance seems to be associated with Zn concentration, as found in other crops, indicating that similar genes are involved in micronutrient loading in seeds and that breeding for both minerals simultaneously is feasible (Blair et al., 2013). Based on QTL localization, Fe and Zn concentration might be associated with the seed storage protein Phaseolin, whereas the Fe storage protein Ferritin was also associated with a QTL (Blair et al., 2009). Indeed, engineering for increased Ferritin expression in endosperm of Poaceae species have been a relatively successful strategy (Sperotto et al., 2012), and thus Ferritin-associated QTLs are interesting candidates. Fe-chelate reductase, which is important for Fe uptake in roots, has also been suggested as a possible candidate gene (Blair et al., 2013). ### WHERE TO FOCUS NEXT? Biofortification for any crop will benefit from multiple approaches, which can improve one another to achieve target Fe seed levels. For common bean, bioavailability tests are key due to the high level of anti-nutrients. The Caco-2 cell in vitro model has been widely used, with better results than previous in vivo absorption models (Ariza-Nieto et al., 2007; Blair et al., 2013; Petry et al., 2016). Recently, a new model using poultry (Gallus gallus) combined with Caco-2 cells showed that they can ### REFERENCES be used as a robust, cost-effective two-step system to evaluate Fe bioavailability, which should be mandatory to generate as well as to monitor biofortified crop seeds after their release (Tako et al., 2016). Another focus should be to independently increase Fe in cotyledons and in seed coat, and understand the physiological roles of phytate/polyphenols and the effects of their reduction on seed viability and seedling establishment. Fe in cotyledons accumulates at the vascular bundles (Cvitanich et al., 2010), similar to Arabidopsis where it depends on Vacuolar Iron Transporter (VIT1; Kim et al., 2006). Phytate is also likely to accumulate in vacuoles, based on the activity of MRP transporters (Nagy et al., 2009; Panzeri et al., 2011). It remains to be determined if the same cells accumulate Fe and phytate, and if the vacuole is the main site where phytatebound Fe is localized. Thus, analyses of cellular and subcellular distribution of Fe and phytate (using phosphorous as a surrogate) will be key for advances in cotyledon biofortification (Punshon et al., 2013). Moreover, understanding how polyphenols affect Fe homeostasis and how their levels could be manipulated will indicate new approaches for seed coat biofortification. ### AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. common bean (Phaseolus vulgaris L.) and association with newly-mapped candidate genes. Mol. Breed. 30, 1265–1277. doi: 10.1007/s11032-012-9713-z acid mutant reveals an exon-excluding splice-site mutation. Theor. Appl. Genet. 125, 1413–1423. doi: 10.1007/s00122-012-1922-7 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Sperotto and Ricachenevsky. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Exogenous Glycine Nitrogen Enhances Accumulation of Glycosylated Flavonoids and Antioxidant Activity in Lettuce (Lactuca sativa L.) Xiao Yang<sup>1</sup> , Xiaoxian Cui 2, 3, Li Zhao<sup>1</sup> , Doudou Guo<sup>1</sup> , Lei Feng<sup>4</sup> , Shiwei Wei <sup>5</sup> , Chao Zhao2, 6 \* and Danfeng Huang<sup>1</sup> \* ### Edited by: Marta Wilton Vasconcelos, Universidade Católica Portuguesa, Portugal #### Reviewed by: Ulrike Mathesius, Australian National University, Australia Francisco A. Tomas-Barberan, Consejo Superior de Investigaciones Científicas (CSIC), Spain #### \Correspondence: Chao Zhao [email protected] Danfeng Huang [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 26 July 2017 Accepted: 24 November 2017 Published: 15 December 2017 #### Citation: Yang X, Cui X, Zhao L, Guo D, Feng L, Wei S, Zhao C and Huang D (2017) Exogenous Glycine Nitrogen Enhances Accumulation of Glycosylated Flavonoids and Antioxidant Activity in Lettuce (Lactuca sativa L.). Front. Plant Sci. 8:2098. doi: 10.3389/fpls.2017.02098 <sup>1</sup> Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China, <sup>2</sup> Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China, <sup>3</sup> Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China, <sup>4</sup> Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, China, <sup>5</sup> Shanghai Agrobiological Gene Center, Shanghai, China, <sup>6</sup> National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China Glycine, the simplest amino acid in nature and one of the most abundant free amino acids in soil, is regarded as a model nutrient in organic nitrogen studies. To date, many studies have focused on the uptake, metabolism and distribution of organic nitrogen in plants, but few have investigated the nutritional performance of plants supplied with organic nitrogen. Lettuce (Lactuca sativa L.), one of the most widely consumed leafy vegetables worldwide, is a significant source of antioxidants and bioactive compounds such as polyphenols, ascorbic acid and tocopherols. In this study, two lettuce cultivars, Shenxuan 1 and Lollo Rossa, were hydroponically cultured in media containing 4.5, 9, or 18 mM glycine or 9 mM nitrate (control) for 4 weeks, and the levels of health-promoting compounds and antioxidant activity of the lettuce leaf extracts were evaluated. Glycine significantly reduced fresh weight compared to control lettuce, while 9 mM glycine significantly increased fresh weight compared to 4.5 or 18 mM glycine. Compared to controls, glycine (18 mM for Shenxuan 1; 9 mM for Lollo Rossa) significantly increased the levels of most antioxidants (including total polyphenols, α-tocopherol) and antioxidant activity, suggesting appropriate glycine supply promotes antioxidant accumulation and activity. Glycine induced most glycosylated quercetin derivatives and luteolin derivatives detected and decreased some phenolic acids compared to nitrate treatment. This study indicates exogenous glycine supplementation could be used strategically to promote the accumulation of health-promoting compounds and antioxidant activity of hydroponically grown lettuce, which could potentially improve human nutrition. Keywords: luteolin, organic nitrogen, nitrate, quercetin, ascorbic acid, H2O2 scavenging capability ### INTRODUCTION A balanced diet is essential to ensure physical development and health. Numerous epidemiological studies have suggested high daily consumption of fruits and vegetables lowers the risk of several chronic diseases, such as cancer, cardiovascular disease and diabetes; the protective effects of fruit and vegetable consumption are mainly attributed to the presence of bioactive phytochemicals such as polyphenols, vitamin C and vitamin E (Arts and Hollman, 2005; Hooper and Cassidy, 2006; Russo et al., 2012; Chen and Chen, 2013; Wang et al., 2017). The economically valuable vegetable crop lettuce (Lactuca sativa) is a minimally processed food product available throughout the entire year, and is a significant source of natural healthpromoting compounds. Multiple factors, such as environmental conditions, agronomical manipulation, harvest time, watering and fertilization can strongly influence the levels of healthpromoting compounds in horticultural plants (Liu et al., 2007; Li and Kubota, 2009; Becker et al., 2014; Tavarini et al., 2015). Specifically, nitrogen fertilization plays an essential role in balancing the yield and quality of edible plants, especially the levels of secondary metabolites. As the classical terrestrial nitrogen cycling paradigm asserts that organic nitrogen must be converted into nitrate or ammonium prior to becoming biologically available, the value of organic nitrogen (especially simple forms, such as amino acids) as a fertilizer has been largely ignored (Ge et al., 2009; Näsholm et al., 2009). Recently, several lines of evidence have suggested organic nitrogen (Ge et al., 2009; Gonzalez-Perez et al., 2015), which represents 96–99% of total nitrogen in soil, can be directly absorbed by plants and significantly influence plant physiology and nutritional quality (Paungfoo-Lonhienne et al., 2008). The simple amino acid glycine is regarded as a proof of life, was the original nutrient form for organisms (Xu et al., 2017) and is one of the most abundant free amino acids in horticultural soil; the glycine concentration of soil ranges from 1.14 to 2.39 µg N/g, corresponding to more than 30% of total free amino acids (Wang et al., 2013; Gonzalez-Perez et al., 2015). Compared to other amino acids, there is lower microbial demand for glycine and it is taken up more rapidly by plants (Lipson et al., 1999). Glycine is regarded as a model amino acid in plant organic nitrogen research. There is growing interest in how nitrogen, especially its inorganic forms, influence antioxidant accumulation and bioactivity. Most studies support the notion that nitrate supply has a negative effect on the biosynthesis of phenolics and vitamin C, as well as antioxidant activity (Lee and Kader, 2000; Awad and de Jager, 2002; Staugaitis et al., 2008; Ibrahim et al., 2012; Yañez-Mansilla et al., 2014). However, recent evidence suggests glycine enhances tolerance to salinity (Badran et al., 2015), drought stress (Yang N. et al., 2016) and cold temperatures (Cao et al., 2017) via elevating the reactive oxygen species (ROS) scavenging system, nitrogen uptake and photosynthesis. In addition, glycine promotes the accumulation of carbohydrates (sucrose, glucose, fructose), which can provide a source of energy and carbon rings for polyphenol biosynthesis (Liu et al., 2016). L-phenylalanine, a flavonoid pathway precursor and phenylalanine ammonia lyase (PAL) substrate, was induced in pak choi by exogenous glycine supply (Wang X. L. et al., 2014). We previously assessed the main changes between lettuce cultured in glycine and nitrate without soil using a non-target metabolomics approach. Glycine nitrogen promoted the accumulation of glycosylated quercetin derivatives and luteolin derivatives (quercetin 3-O-glucoside, quercetin 3-O-malonylglucoside, luteolin 7-O-glucoside, and luteolin 7-O-glucronide), ascorbic acid and amino acids, but reduced the levels of some phenolic acid derivatives and some organic acids involved in the tricarboxylic acid cycle (Yang et al., 2018). Luteolin 7-O and quercetin 3-O glycosides are potent free radical scavengers/antioxidants and prevent ROS generation effectively (Agati et al., 2012; Brunetti et al., 2013). Therefore, we hypothesized glycine supply could promote the synthesis of health-promoting compounds in lettuce. Thus, in the present study, the influence of different concentrations of organic nitrogen (as glycine) on the nutritional quality (i.e., total polyphenol, flavone, vitamin C and vitamin E contents, and antioxidative activity) of two lettuce cultivars was determined using a metabolomics approach and in vitro bioactivity assays. This work further explores the biological effects of organic nitrogen supply and indicates exogenous supply of glycine could potentially be used to enhance the nutritional quality of lettuce. ### MATERIALS AND METHODS ### Plants and Cultivation Seeds of the lettuce cv. Lollo Rossa and cv. Shenxuan 1 were purchased from Atlas Seeds BJ Co., Ltd, (Beijing, China) and the Horticultural Research Institute, Shanghai Academy of Agriculture Sciences (China), respectively. Seeds were sown in white mesh pot net baskets (Figure 1) in nursing substrate (100% perlite) and germinated in a greenhouse at Shanghai Sunqiao Modern Agricultural Development Zone, China (latitude 31◦ 17′ N, longitude 121◦ 62′ E; altitude, 4 m above mean sea level). Then, 21-day-old seedlings were transferred to a water-cycled hydroponic experiment device (Figure 1). After recovering the seedlings in water for 3 days, the seedlings were cultivated for 30 days in nitrogenous nutrient solution (1.25 mM Mg, 3.5 mM K, 1.25 mM S, 2.05 mM Ca, 1 mM P, and 6.6 mM Cl, pH 5.8) containing different forms and concentrations of nitrogen: 9 mM nitrate (as NaNO3, control, 9Nit), 4.5 mM glycine (4.5Gly), 9 mM glycine (9Gly) or 18 mM glycine (18Gly). Ampicillin (10 mg/L) was added to the nutrient solution to prevent bacterial infections (Okamoto and Okada, 2004). All treatments (90 seedlings per treatment, three replicates) were arranged randomly. During the experiment, environmental conditions were maintained at 22 ± 3 ◦C during the day and 15 ± 2 ◦C at night with 250–280 µmol·m−<sup>2</sup> ·s <sup>−</sup><sup>1</sup> during the 14 h photoperiod (natural and artificial lighting). The culture solutions were contained in a circulating water system and renewed every 2 days. At the end of cultivation, all leaves were collected. Each sample was divided in two: one half was used for physiological assessments; the other half was flash frozen in liquid nitrogen and stored at −80◦C until further analysis. ### Chemicals and Reagents Ultra-pure water was prepared using a Milli-Q system (Millipore Laboratory, Bedford, MA, USA). Methanol and acetonitrile (LC-MS grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA); luteolin 7-glucoside, quercetin glucoside and chicoric acid standards (HPLC grade, ≥ 98%), from the Chinese National Institute for Food and Drug Control (Beijing, China); methoxyamine hydrochloride, L-2-chlorophenylalanine, bis (trimethylsilyl) trifluoroacetamide (BSTFA), and 2′ ,7′ dichlorofluorescin diacetate (DCFH-DA), from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum, from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA); L-glutamine and penicillin, from Sangon Biotech (Shanghai, China); the Cell Counting Kit-8, from Dojindo (Kumamoto, Japan); and 2, 2′ -azobis (2-amidinopropane) dihydrochloride (ABAP), form Wako (Osaka, Japan). All other chemicals were of analytical grade and obtained from China National Medicines Co., Ltd. (Shanghai, China). ### Analysis of the Fresh Weight and Total Phenolic and Anthocyanin Contents of Lettuce Leaves Fresh weight was measured using electronic scales (AUY220; Shimadzu, Kyoto, Japan). For total phenolic content analysis, 1 g of raw leaf was ground in 10 mL of 0.1 mM HCl-methanol (v/v 1:1), extracted ultrasonically for 30 min and centrifuged at 9,000 g for 30 min. The supernatant was diluted to 25 mL with methanol and filtered through a 0.45µm membrane (Złotek et al., 2014). Total polyphenol content was quantified using a UV-Vis spectrophotometer (U2900; Hitachi, Tokyo, Japan) at 725 nm as described by Złotek et al. (2014) and expressed as mg gallic acid equivalent (GAE) per g fresh weight (mg GAE·g <sup>−</sup><sup>1</sup> FW). The formula for calculating GEA was Y (GAE, mg) = 0.1266 × (OD725nm) − 0.0008. Total anthocyanin content was measured via a nondestructive method, as described previously (Yang X. et al., 2016; Ferrandino et al., 2017), using a Dualex 4 Scientific<sup>+</sup> (Dx4, FORCE-A, Orsay, France) and expressed as: anthocyanin content (ng per cm<sup>2</sup> ) = log (red-excited infrared fluorescence/greenexcited infrared fluorescence) × 10<sup>3</sup> . ### UPLC-VION-IMS-QTOF-MS/MS Analysis For UPLC-MS, lettuce leaf samples (200 mg) were weighed, ground into a powder in liquid nitrogen, extracted in 1 mL TABLE 1 \| Plant fresh and dry weights and total anthocyanin contents of lettuce cultivated in hydroponic solution containing nitrate or glycine. For fresh weight analysis, values are means ± SD (n = 4); for total anthocyanidin content, values are mean ± SD (n = 100); Value with different letters are significantly different; p < 0.05, LSD analysis. n.d. represents not detected. TABLE 2 \| Metabolites putatively identified by UHPLC-IMS-QTOF-MS in the leaf extracts of nitrate- and glycine-treated lettuce. (Continued) ### TABLE 2 \| Continued RT, Retention time. <sup>a</sup>5-Caffeoylquinic acid and 3-caffeoylquinic acid were the major forms. <sup>b</sup>3,5-Di-O-caffeoylquinic acid was reported as the major form. methanol/water (80:20, v/v), sonicated at 25◦C for 30 min, incubated at 4◦C for 12 h, centrifuged at 12,000 g for 10 min, and 0.5 mL supernatant was used for UPLC-MS analysis as previously described (Abu-Reidah et al., 2013a; Yang et al., 2018). The composition and relative contents of polyphenols in lettuce leaves were analyzed using an Acquity class UPLC and Vion IMS QTOF MS (Waters, Corp. Milford, MA, USA) using an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, i,d.: 1.7µm). The mobile phases were water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The injection volume was 3 µL, flow rate was 0.4 mL/min, with gradient elution (0–4 min, 20% B; 4–6 min, linear gradient from 20 to 25% B; 6– 8.5 min, 50% B; 8.5–12.5 min 50–85% B, 12.5–14 min, 85–100% B; 14–17 min, 100%), then initial conditions were restored for 5 min to equilibrate the column. The scan range was 50–1,000 m/z, and spectra were acquired in negative-ion mode. MS and MS/MS spectra were identified based on accurate mass, MS<sup>2</sup> fragments and isotopic distribution using online databases (e.g., ResPect, http://spectra.psc.riken.jp/) and bibliographies related to lettuce metabolites. MS and MS/MS tolerance were set at 3 mDa and 10 mDa, respectively. MS and MS/MS data were processed using Progenesis QI software (Waters Corp.). To evaluate analytical reliability and reproducibly, quality control (QC) samples (a mixture of all samples) were analyzed at the start, middle and end of each batch, as previously described (Want et al., 2013). Principal component analysis (PCA) showed the PCA scores of the seven QC samples clustered together (Supplemental Figure 1), confirming the reliability and repeatability of the metabolomic analysis. ### GC-MS Analysis For GC-MS analysis, 200 mg lettuce leaf tissue was ground in liquid N, extracted in 1 mL ice-cold methanol:chloroform (3:1 v/v), 20 µL of 0.3 mg·mL−<sup>1</sup> L-2-chlorophenylalanine (internal standard) was added, the samples were centrifuged at 15,000 g for 10 min, 0.3 mL of the supernatant was vacuum freeze-dried at 25◦C, 80 µL methoxyamine hydrochloride (15 mg mL−<sup>1</sup> in pyridine) was added, incubated at 37◦C for 1.5 h, then 80 µL bis (trimethylsilyl) trifluoroacetamide (BSTFA, containing 1% trimethylchlorosilane) was added and incubated at 80◦C for 1 h (Du et al., 2011). Relative GC/MS quantification of ascorbic acid, α-tocopherol and γ-tocopherol were performed using an Agilent 7890 Gas Chromatograph coupled to a LECO Mass Spectrometer (PerkinElmer Inc., Waltham, MA, USA) using a DB-5MS capillary column (30 m × 0.25 mm × 0.25µm; Agilent J&W Scientific, Folsom, CA, USA). Inlet temperature, transfer line temperature and ion source temperature were 280◦C, 280◦C, and 230◦C, respectively. The gas (helium) flow rate was 1 mL·min−<sup>1</sup> and injection volume was 1 µL. After 6.5 min solvent delay, the initial GC oven temperature was 60◦C; 1 min after injection, the GC oven temperature was raised to 300◦C at 5◦C/min, then held at 300◦C for 11 min. Measurements were made via electron impact ionization (70 eV) in full scan mode (m/z 33–600). Ascorbic acid, α-tocopherol and γ-tocopherol were identified using LECO Chroma TOF (PerkinElmer Inc.) by comparison with reference spectra in the NIST 14 Mass Spectral Library (Scientific Instrument Services, Inc. NJ, USA). Relative response ratio was obtained by dividing the peak area by the peak area for L-2 chlorophenylalanine. ### Antioxidant Bioactivity Analysis Reducing Potential Assay The antioxidant activity of the lettuce leaf extracts was determined using the ferric-reducing antioxidant power assay (FRAP) (Chan et al., 2007). Samples (2.5 mL, extracted as described for polyphenol analysis) were mixed with 2.5 mL phosphate buffer (0.2 mM, pH 6.6) and 2.5 mL potassium ferricyanide (1% w/v), incubated at 50◦C for 20 min, and then immediately transferred onto ice. Trichloroacetic acid solution (2.5 mL of 10% w/v) was added to stop the reaction, the mixture was centrifuged at 3,000 g for 10 min, then 2.5 mL of the supernatant was diluted with 2.5 mL water, 0.5 mL ferric chloride solution (0.1% w/v) was added, incubated for 30 min and absorbance was determined at 700 nm. The extraction solution used for polyphenol analysis was used as control sample. FRAP values were expressed as mg GAE·g <sup>−</sup><sup>1</sup> FW. ### Preparation of Extracts for Cellular Antioxidant Activity Assays Lettuce leaf extracts were prepared as described for UPLC-MS analysis. Prior to the antioxidant bioactivity assays, 0.5 mL of supernatant from each sample was vacuum freeze-dried at 25◦C and resuspended in 200 µL water containing 0.1% DMSO. ### Cytotoxicity Assay Hepatitis B virus-producing HepG2 cells were cultured in DMEM supplemented with 2 mM L-glutamine, 50 U mL−<sup>1</sup> penicillin and 10% fetal bovine serum at 37◦C in a 5% CO<sup>2</sup> atmosphere (Hong et al., 2013). The cytotoxicity of the lettuce leaf extracts toward HepG2 cells was assessed using the CCK-8 assay, as described by Shi et al. (2017). Cell viability (%) was calculated as (OD450 (sample) – OD450 (blank))/(OD (mock) – OD450 (blank)). ### Cellular Antioxidant Activity (CAA) Assay Cellular antioxidant activity (CAA) was quantified as previously described (Wolfe and Liu, 2007). Briefly, HepG2 cells were seeded at a density of 1 × 10<sup>5</sup> cells per well into 96-well microplates in 100 µL media. After 24 h, the media was removed, cells were washed with PBS, then incubated with 100 µL of media containing 25µM DCFH-DA and 0.5 µL lettuce leaf extract for 1 h at 37◦C. Then the solution was removed, 100 µL of 600µM ABAP was added, and fluorescence values were read at 485 nm excitation and 538 nm emission using a VictorTM X3 Multilabel Plate Reader (Perkin Elmer) every 5 min for 1 h. CAA was expressed as CAA (unit) =100 – (R SA /R CA) × 100, where SA is the integrated area of sample fluorescence vs. time curve and CA is the integrated area of the control curve. ### Cellular H2O<sup>2</sup> Scavenging Assay HepG2 cells were seeded at 5,000 cells/well in 96-well plates in 100 µL media, cultured for 24 h, incubated with 400µM H2O<sup>2</sup> containing 1 µL of lettuce leaf extract or an equivalent volume of media (mock) or H2O<sup>2</sup> (400µM H2O<sup>2</sup> solution plus 1 µL medium) as control treatments for 24 h, and cell viability was assessed using the CCK-8 assay as described by Shi et al. (2017). ### Statistical Analysis Values are the mean ± SD of three biological replicates per treatment and three technical replicates per sample. ANOVA based on LSD analysis and Students t-tests were performed using IBM SPSS Statistics 22 (IBM, Armonk, NY, USA); p < 0.05 was considered significant. Pathway analysis was performed using ProcessOn (https://www.processon.com/) and R software (https://www.r-project.org/). PCA analysis was performed using SIMCA-P13.0 (Sartorius Stedim Biotech, Gottingen, Germany), Pearson correlation analysis was conducted using R software. Figures were created using R software or OriginPro 2016 (OriginLab, Northampton, MA, USA). ### RESULTS ### Effect of Glycine on Growth of Lettuce The fresh weights of the aboveground parts and whole lettuce plants after 30 days cultivation in hydroponic solution containing 9 mM nitrate (control) or 4.5, 9, or 18 mM glycine are shown in Table 1. Glycine significantly reduced the fresh and aboveground weights compared to control lettuce. Among the glycine-treated plants, 9 mM glycine led to a significantly higher fresh weight (p < 0.05) than 4.5 or 18 mM glycine. ### Effect of Glycine on Accumulation of Antioxidative Compounds In this study, the total anthocyanidin content was only assessed in the Lollo Rossa cultivar; the Shenxuan 1 cultivar is a green leafy lettuce, which does not contain detectable levels of anthocyanidins. As shown in Table 1, glycine supply increased (p < 0.05) the total anthocyanidin content in Lollo Rossa leaves compared to control plants. The anthocyanidin level peaked at 34.72 ng per cm<sup>2</sup> in Lollo Rossa leaves exposed to 18 mM glycine, which was significantly higher than the plants treated by 4.5 or 9 mM glycine. The highest exogenous concentration of glycine (18 mM) also significantly (p < 0.05) increased the total polyphenol content of the lettuce leaves compared to lettuce cultivated in 9 mM nitrate or 4.5 or 9 mM glycine (Figure 2A), with maximal levels of 1.48 and 2.53 mg g−<sup>1</sup> observed in Shenxuan 1 and Lollo Rossa, respectively. In addition, relative ascorbic acid content increased significantly as glycine supply increased (from 4.5 to 9 mM glycine), but decreased at 18 mM glycine (Figure 2B) compared to control lettuce. The α-tocopherol content peaked in lettuce exposed to 18 mM glycine, corresponding to respective 3.4- and 1.7-fold increases in Shenxuan 1 and Lollo Rossa compared to the controls supplied with 9 mM nitrate (Figure 2C). Moreover, in both varieties, the levels of γ-tocopherol were significantly higher in lettuce supplied with 4.5 and 18 mM glycine (p < 0.05) than control plants (Figure 2D). UPLC-MS analysis can separate co-effluents and enables robust and reproducible identification of the isomeric structures of polar metabolites (e.g., phenolic compounds) (Paglia et al., 2014). By comparison with online and in-house databases as well as published data, a total of 35 polyphenols were tentatively identified in the lettuce leaf extracts (level 2, putatively-annotated compounds), including 17 phenolic acid derivatives and 18 glycosylated flavonoids (Table 2). Metabolic pathway analysis was conducted to investigate the relationships between glycine supply and the accumulation of phenolic acids and flavonoids (Figure 3). As shown in Figures 4, 5, the relative contents of apigenin 7-O, luteolin 7-O and quercetin 3-O and caffeoylquinic acids derivatives in Lollo Rossa cultivar are higher than their in Shenxuan 1 lettuce. In the Shenxuan 1 cultivar, glycine supply significantly decreased the contents of dihydroxybenzoic acid derivatives (dihydroxybenzoic acid hexoside isomer 1 and 2), p-coumaroylquinic acid, dihydrocaffeic acid sulfate, tri-4-hydroxyphenylacetyl glucoside, ferulic acid methyl ester, caffeoyl hexose, hydroxybenzoyl dihydroxybenzoyl hexose and syringic acid hexose compared to control lettuce cultivated in nitrate; all of these metabolites were present at the highest levels in lettuce cultivated in 4.5 or 18 mM glycine and lowest levels in lettuce cultivated in 9 mM glycine (Figure 4). In addition, apigenin 7-O-glucuronide and some luteolin glycoside derivatives (luteolin 7-O-glucoside, luteolin 7-glucuronide) and quercetin glycoside derivatives (quercetin 3-glucuronide, quercetin 3-O-(6′′-O-malonyl)-glucoside 7-O-glucuronide, quercetin 3-O-(6′′ -O-malonyl)-glucoside 7-O-glucoside, quercetin glucose acetate isomer 1 and 2) were not detected in control Shenxuan 1 lettuce, but were induced by 9 and 18 mM glycine (Figure 5). In the Lollo Rossa cultivar, glycosylated luteolin derivatives, quercetin derivatives, apigenin derivatives, dihydrocaffeic acid derivatives (dihydrocaffeic acid hexose isomer 2, dihydrocaffeic acid sulfate), tri-4-hydroxyphenylacetyl glucoside, syringic acid hexose, ferulic acid methyl ester, syringaresinol glucoside and esculetin hexoside were significantly induced by glycine, whereas the dihydroxybenzoic acid derivatives (dihydroxybenzoic acid hexose isomer 1 and 2) and hydroxybenzoyl dihydroxybenzoyl hexose were significantly reduced by 9 and 18 mM glycine (Figures 4, 5). ### Effect of Glycine on Antioxidant Activity To assess the effect of glycine supply on antioxidant activity, in vitro assays were performed to directly evaluate simple ferric reducing ability and cellular antioxidant activity (Figures 6A,B). Both nitrate- and glycine-treated lettuce leaf extracts had low cytotoxicity toward HepG2 cells in the CCK-8 assay (Figure 6C). However, supply of 9 or 18 mM glycine significantly increased the antioxidant activity of the Lollo Rossa cultivar extracts, FIGURE 3 \| Effect of glycine and nitrate supply on the composition and concentrations of polyphenols in lettuce leaf extracts. Relative abundance of metabolites is indicated from red (high) to green (low). The dotted lines in the metabolic pathway represent possible relationships that have not yet been proven experimentally and solid lines indicate pathways in the KEGG or PlantCyc databases. which peaked in lettuce supplied with 18 mM glycine with a 3.3 fold (CAA) and 1.93-fold (FRAP) increase compared to control plants. In the Shenxuan 1 cultivar, 18 mM glycine significantly increased FRAP by 1.4-fold and CAA by 1.8-fold compared to nitrate-treated control plants (Figures 6A,B). Moreover, the CCK-8 assay was conducted to evaluate the viability of HepG2 cells and verify the effect of extraction on H2O<sup>2</sup> scavenging capability (Figure 7). Cell viability was significantly higher in all treatments compared to H2O<sup>2</sup> treatment, suggesting the extraction process is superoxidescavenging. In addition, no significant differences were observed between the mock treatments and all Lollo Rossa samples supplied with glycine and Shenxuan 1 samples supplied with 18 mM glycine. Glycine-treated lettuce exhibited higher scavenging capability than nitrate-treated control lettuce. In the Shenxuan 1 cultivar, the cell viability of lettuce exposed to 18 mM glycine was significantly higher than control lettuce, while the extracts of lettuce treated with 4.5 and 9 mM glycine increased cell viability compared to the 9 mM nitrate-treated extracts. ### DISCUSSION ### Glycine Nitrogen Supply Reduces the Fresh Weight of Lettuce Plants The ability of higher plants to use organic nitrogen (amino acids, peptides and proteins) as a nitrogen source has been demonstrated in laboratory studies and field experiments (Näsholm et al., 2009). Compared to nitrogen deficiency, exogenous glycine supply increases production of biomass in Arabidopsis plants (Forsum et al., 2008). However, glycine could not support plant growth to the same extent as the same concentration of nitrate in pak choi (Wang, X. et al., 2014). In this study, glycine nitrogen supply significantly decreased the fresh and dry weight of lettuce compared to plants supplied with 9 mM nitrate. In horticulture, 9 mM is suggested as a standard reference concentration in commercial hydroponic lettuce production (Brechner and Both, 2017, Grower's Handbook: Lettuce). It is not surprising that glycine supply decreases plant growth compared to plants provided with the appropriate nitrate concentration in agricultural practice. These results are in accordance with our previous studies of pak choi (Wang X. et al., 2014), which indicated glycine may limit plant root growth (Dominguez-May et al., 2013) and induce differential proteomic responses associated with plant defense or stress and energy and nitrogen metabolism (Wang X. et al., 2014). ### Appropriate Concentrations of Glycine Promote Accumulation of Antioxidants Primary antioxidants, such as flavonoids, ascorbic, acid and tocopherols, are abundant in plants, exert various physiological functions (Dixon et al., 2002; Singh and Singh, 2008) and play significant roles in the human diet (Chen and Chen, 2013; Tomas-Barberan et al., 2016). In this study, 18 mM glycine supply significantly (p < 0.05) increased total polyphenols and anthocyanidin content compared to 9 mM nitrate or lower concentrations of glycine (4.5 or 9 mM). The influence of nitrogen on the accumulation and bioactivity of antioxidants in plants are controversial. Generally, high levels of inorganic nitrogen (nitrate or ammonium) exert negative, dose-dependent effects on plant flavonoid biosynthesis and anthocyanin accumulation and activity (Patil and Alva, 1999; Awad and de Jager, 2002; Yañez-Mansilla et al., 2014; Becker et al., 2015). In addition to environmental conditions (e.g., temperature, light), the dose-dependent variations observed in plants exposed to different nitrogen sources may also be due to the complexity of plant responses to nutrient availability. According to the growthdifferentiation balance hypothesis, plants with sufficient nitrogen supply (e.g., 9 mM nitrate in this study) tend to allocate nitrogen to vegetative growth rather than biosynthesis of phenolics; low supply (e.g., 4.5 mM glycine) limits both growth and secondary metabolite accumulation, In contrast, plants with intermediate resources (e.g., 9 or 18 mM glycine, as N use efficiency is lower for glycine compared with nitrate) accumulate high levels of phenolic acids with an intermediate increase in biomass (Herms and Mattson, 1992; Glynn et al., 2007). Ascorbic acid is a major antioxidant in lettuce (Nicolle et al., 2004). In this study, ascorbic acid was significantly induced by 9 mM glycine compared to control nitrate and 4.5 or 18 mM glycine. The effects of different concentrations of nitrogen on ascorbic acid synthesis remain controversial. Some studies have indicated increased nitrogen supply increases the vitamin C content in some plants, though most studies reported ascorbic acid decreased or did not significantly change (see review by Mozafar, 1993; Flores et al., 2004). This controversy may be related to inter-plant variations in the optimum nitrogen concentration required for maximal vitamin C accumulation. Tocopherol is a lipid-soluble natural antioxidant; the α- and γforms are the major isomers in lettuce (Nicolle et al., 2004; Cruz et al., 2014). Exposure to 18 mM glycine significantly increased the α-tocopherol content compared to lettuce cultivated in 9 mM nitrate and 4.5 or 9 mM glycine. In both varieties of lettuce, the concentration of γ-tocopherol was significantly higher for plants supplied with 4.5 and 18 mM glycine than control plants. Similarly, previous research reported inorganic nitrogen fertilization increased the concentration of tocopherols in rapeseed, increasing the levels of urea more than the levels of ammonium (Hussain et al., 2014). ### Glycine Supply Promotes Accumulation of Apigenin-3-O, Quercetin-3-O and Luteolin-7-O Glycoside Derivatives The phenylpropanoid and flavonoid pathways synthesize a wide range of secondary metabolites including phenolic acid derivatives, lignins and flavonoids, which play important roles in both plant growth and human nutrition (Tzin and Galili, 2010). Glycine supply decreased the contents of several phenolic acids (e.g., hydroxycinnamic and hydroxybenzoic derivatives in the Shenxuan 1 cultivar; hydroxybenzoic derivatives in Lollo Rossa), but led to accumulation of luteolin, apigenin and quercetin glycoside derivatives. In general, phenolic acid derivatives and flavonoid biosynthesis share the same precursor, p-coumaroyl CoA. The induction of flavonoid biosynthesis and reductions in the content of some phenolic acids and derivatives observed in the presence of glycine indicate altered precursor availability induced metabolic flux from phenolic acid biosynthesis to flavonoid pathways by altering the expression of chalcone synthesis and auxin polar transport (Besseau et al., 2007; Taulavuori et al., 2016). In addition, glycine significantly promoted the accumulation of sugars, which may positively stimulate the biosynthesis of flavonol glycosides by increasing the supply of carbon rings and glycosides (Liu et al., 2016). Luteolin and quercetin derivatives have a greater capacity to scavenge ROS than most other flavonoids (Brunetti et al., 2013), thus an increase in the luteolin to apigenin glycosides ratio and kaempferol to quercetin glycosides ratio are a component of plant responses to light quality and intensity; luteolin (or quercetin) glycoside derivatives increased significantly, while apigenin (or kaempferol) glycosides derivatives increased only slightly in response to light (Markham et al., 1998; Tegelberg and Julkunen-Tiitto, 2001; Oh et al., 2009). In this study, apigenin glycosides were not detected (Shenxuan 1) or only present at trace levels (Lollo Rossa) in the control lettuce supplied with nitrate, whereas a high concentration of glycine (18 mM) induced accumulation of apigenin glycosides. In addition, the downstream metabolites luteolin glycoside derivatives and another dihydroxy B-ring-substituted flavonoid (quercetin 3-O glycoside derivatives) were also significantly induced by glycine compared to control lettuce. For example, 15-fold (Lollo Rossa) and 2-fold (Shenxuan 1) increases in luteolin 7-glucuronide were observed in lettuce supplied with 18 mM glycine compared to the respective control lettuce supplied with 9 mM nitrate. Moreover, 10- and 3-fold increases in quercetin glucoside were observed in Lollo Rossa and Shenxuan 1 supplied with 18 mM glycine compared to control lettuce. Thus, we hypothesize that apigenin-3-O, quercetin-3-O, and luteolin-7-O glycoside derivatives may represent signals of the response to glycine supply and indicate a metabolic switch from accumulation of small quantities of glycosylated flavonoids to synthesis of both monohydroxy and dihydroxy B-ring-substituted flavonoid derivatives. ### Appropriate Concentrations of Glycine Promote Antioxidant Bioactivity Genotype and growing conditions influence antioxidant compositions and bioactivity in lettuce. Red leafed lettuce cultivars have higher average total polyphenol contents and antioxidant capacities than green leafed cultivars (Liu et al., 2007). In this study, extracts from the Lollo Rossa cultivar exhibited significantly stronger ferric-reducing antioxidant power, cellular antioxidant activity and H2O<sup>2</sup> scavenging ability than the Shenxuan 1 cultivar. The Lollo Rossa cultivar is likely to contain significantly higher levels of polyphenols (particularly glycosylated quercetin, apigenin, and luteolin), vitamin C and anthocyanins, which correlate positively with antioxidative activity. We performed Pearson Correlation analysis to investigate the possibility of an inter-relationship between the metabolites detected and antioxidant activity, as indicated by FRAP, CAA, and H2O<sup>2</sup> scavenging capability (Figure 8). Antioxidant bioactivity was significantly (p < 0.05) and positively (r > 0.75) correlated with total polyphenol content and the levels of apigenin 7-O-glucuronide, luteolin 7-glucoside, quercetin 3-O-(6′′ -O-malonyl)-glucoside 7-O-glucoside, quercetin 3-O- (6′′ -O-malonyl)-glucoside 7-O-glucuronide, quercetin glucose acetate isomer 2, quercetin glucoside and quercetin hexoside glucuronide. These results are in agreement with a previous study of Stevia rebaudiana leaves treated with nitrogen, which found antioxidant bioactivity positively correlated with total phenolic acids and the levels of glycosylated quercetin, apigenin, and luteolin (Tavarini et al., 2015). Glycine-treated lettuce extracts exhibited higher scavenging capability than nitrate-treated control lettuce extracts. The antioxidant bioactivity of Shenxuan 1 lettuce exposed to 18 mM glycine was significantly higher than that of control lettuce, while the extracts of lettuce treated with 9 and 18 mM glycine had higher antioxidative activities than the 9 mM nitratetreated extracts. These results can mainly be attributed to the significantly higher total levels of polyphenols, particularly luteolin, quercetin and apigenin glycosides, in the lettuce treated with 18 mM glycine. A luteolin or quercetin-rich diet is related to reduced risks of specific types of cancer (Ekström et al., 2010; Lam et al., 2012; Lin et al., 2014) and cardiovascular disease (Duthie et al., 2000; Lee et al., 2011), and plays a protective effect in diabetes (Babu et al., 2013). Thus, exogenous glycine supply may promote the accumulation of healthpromoting compounds and increase the antioxidative activity of lettuce, which could potentially be beneficial for human nutrition. ### CONCLUSION The appropriate concentration of glycine (18 mM for Shenxuan 1; 9 mM for Lollo Rossa) significantly enhanced the levels of antioxidants, including total polyphenols and α-tocopherol, and antioxidant activity (as indicated by FRAP, CAA, and H2O<sup>2</sup> scavenging capability) compared to lettuce supplied with nitrate. Most glycosylated flavonoids detected, including apigenin, quercetin and luteolin, were also induced by 9 and 18 mM glycine, whereas glycine decreased the levels of some phenolic acids. This study indicates exogenous glycine supply could be used strategically to promote the accumulation of health-promoting compounds and increase the antioxidative activity of hydroponically grown lettuce; this strategy may have potential relevance to human nutrition. ## AUTHOR CONTRIBUTIONS XY, XC, and LZ performed all the experimental measurements, analyzed the data, and drafted the manuscript. DG, LF, and SW helped with the figures and samples. CZ and DH designed experiment and supervised all the results, and contributed to writing the manuscript. ### FUNDING This work was supported by the National Natural Science Foundation of China (No. 61233006 and No. 81370046); the Seed Industry Development Project of Shanghai, China (Grant No. 2016, 1-8); and the Agriculture Research System of Shanghai, China (Grant No. 201702). ### ACKNOWLEDGMENTS We acknowledge lab members Miss. Yifei Zhao, Miss. Yanwen Gu, and Mr. Jiaxin Zheng for assisting with the ### REFERENCES experiments, and colleague Dr. Bin Liu, Mr. Muhammad Khalid, Mr. Kai Dou, Dr. Xiaosong Liu (Chinese Academy of Sciences), and Mr. Hongkai Zhu (University of Copenhagen) for providing some advice in preparation of the manuscript. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017. 02098/full#supplementary-material to enhanced ultraviolet-B radiation. Physiol. Plant. 113, 541–547. doi: 10.1034/j.1399-3054.2001.1130413.x Conflict of Interest Statement:* The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Yang, Cui, Zhao, Guo, Feng, Wei, Zhao and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Genome-Wide Identification and Expression Analysis of NRAMP Family Genes in Soybean (Glycine Max L.) Lu Qin1†, Peipei Han1†, Liyu Chen<sup>2</sup> , Thomas C. Walk <sup>3</sup> , Yinshui Li <sup>1</sup> , Xiaojia Hu<sup>1</sup> , Lihua Xie<sup>1</sup> , Hong Liao<sup>2</sup> and Xing Liao<sup>1</sup> \* <sup>1</sup> Key Laboratory of Biology and Genetics Improvement of Oil Crops of the Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan, China, <sup>2</sup> Root Biology Center, Fujian Agriculture and Forestry University, Fuzhou, China, <sup>3</sup> Golden Fidelity LLC, St. Louis, MO, United States ### Edited by: Raul Antonio Sperotto, Centro Universitário Univates, Brazil #### Reviewed by: Jitender Giri, National Institute of Plant Genome Research, India Rupesh Kailasrao Deshmukh, Laval University, Canada Victoria Fernandez, Universidad Politécnica de Madrid (UPM), Spain > \Correspondence: Xing Liao [email protected] † These authors have contributed equally to this work. ### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 09 April 2017 Accepted: 03 August 2017 Published: 18 August 2017 #### Citation: Qin L, Han P, Chen L, Walk TC, Li Y, Hu X, Xie L, Liao H and Liao X (2017) Genome-Wide Identification and Expression Analysis of NRAMP Family Genes in Soybean (Glycine Max L.). Front. Plant Sci. 8:1436. doi: 10.3389/fpls.2017.01436 The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis. Keywords: soybean, NRAMP gene family, nutrient deficiency, divalent metal toxicity, nodules ### INTRODUCTION Iron (Fe) is an essential element for plant development and growth, with functions in several basic cellular processes, including photosynthesis, respiration, and chlorophyll biosynthesis (Kobayashi and Nishizawa, 2012). Furthermore, Fe is also a vital component in heme, the Fe-sulfur (S) cluster, and other Fe-binding sites (Kobayashi and Nishizawa, 2012). Given these requirements and Fe deficiency is common in soils, plants have evolved highly efficient systems to acquire 350* Fe from the soil (Kim and Guerinot, 2007). Furthermore, the uptake, utilization, and storage of Fe are also tightly controlled by the coordination of multiple mechanisms regulated to at the transcriptional and post-translational levels (Kobayashi and Nishizawa, 2012). Mechanisms contributing to Fe acquisition in a number of plant species can be divided into two categories (Hell and Stephan, 2003; Morrissey and Guerinot, 2009; Conte and Walker, 2011). Strategy I, which is found in non-graminaceous plants, utilizes IRT1 as the primary transporter responsible for uptake of Fe from soil into roots (Eide et al., 1996; Hell and Stephan, 2003; Walker and Connolly, 2008). Meanwhile, YSL is the main transporter responsible for uptake of Fe from siderophore-Fe complexes into Strategy II graminaceous plants (Curie et al., 2001; Inoue et al., 2009; Thomine and Vert, 2013). Beyond these Strategy I and II transporters, the NRAMP family represents another transporter family associated with Fe uptake and transport (Thomine and Vert, 2013). The NRAMP family, with its highly conserved domain, is widespread in genomes ranging from bacteria to humans (Nevo and Nelson, 2006). It is known to mediate transport of divalent metal ions, such as Fe and manganese (Mn) across cellular membranes. The first known NRAMP protein (NRAMP1) was discovered in mice phagosomal membranes, and was found to function in natural defense against infections by intracellular parasites (Vidal et al., 1993). In contrast to NRAMP1, mice NRAMP2 (also called DMT1), yet it still acts as a divalent metal ion transporter in the absorption of Fe, Mn, zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb) (Garrick et al., 2006). Mutations in NRAMP2 have been associated with defects in Fe absorption and result in Microcytic anemia in mice and the anemic Belgrade rat (Fleming et al., 1998). NRAMP homologs with similar function also were found in human (Cellier et al., 1994; Beaumont et al., 2006; Illing et al., 2012). Several NRAMP gene family members have also been functionally characterized in plants. In Arabidopsis, there are six NRAMP proteins (Mäser et al., 2001). AtNRAMP1 regulates Fe homoeostasis (Curie et al., 2000), and function as a high-affinity transporter for Mn uptake (Cailliatte et al., 2010). AtNRAMP3 and AtNRAMP4 are both localized on the vacuolar membrane and participate in vacuolar Fe mobilization during seed germination (Lanquar et al., 2005). AtNRAMP6 is targeted to a vesicular-shaped endomembrane compartment and functions as an intracellular metal transporter, with possible involvement in Cd tolerance (Cailliatte et al., 2009). In rice, three NRAMP proteins participate in Fe, Cd, and Mn uptake (Takahashi et al., 2011; Sasaki et al., 2012; Yang et al., 2014), while another, OsNrat1, encodes a transporter mediating aluminum (Al) uptake from root tip cell walls into the cell, which contributes to rice Al tolerance (Li et al., 2014). In recent years, several NRAMP genes have been identified in legumes. For example, a peanut NRAMP gene, AhNRAMP1, is significantly induced by Fe deficiency in roots and leaves, and heterologous expression of AhNRAMP1 in tobacco leads to Fe accumulation in young leaves and tolerance to Fe deprivation (Xiong et al., 2012). Moreover, in the model legume Medicago truncatula, MtNRAMP1, is mainly localized to the plasma membrane, with expression levels highest in roots and nodules, suggesting it was the major transporter responsible for apoplastic Fe uptake in rhizobia-infected cells (Tejada-Jiménez et al., 2015). Provided the commercial significance of Soybean (Glycine max L.) worldwide and the detrimental effects of Fe deficiency on yield and quality, it is key to improve our understanding of Fe transport as tool for improving soybean Fe utilization. However, little data is available concerning the NRAMP gene family in soybean until now. In the present study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes. Subsequently, tissue-specific expression of GmNRAMP genes and their responses to various nutrient stresses were all analyzed. The genome-wide analysis of soybean NRAMP genes herein provides a basis to further investigate detailed functions of NRAMP genes in soybean. ### MATERIALS AND METHODS ### Identification and Bioinformatics Analyses of NRAMP Genes in Soybean To identify NRAMP homologs in soybean, nucleic acid, and amino sequences of all reported NRAMPs in Arabidopsis, Rice and Medicago, were used as query sequences in BLASTN (Target type: Genome) and BLASTP (Target type: Proteome) searches of the G. max cultivar Williams 82 in the Phytozome genome database (https://phytozome.jgi.doe.gov/pz/portal.html#) using default settings for E-value and the number of hit sequences. Then, all returned genes and proteins were further examined for inclusion of the conserved Nramp domain (PF01566) by querying in the Uniprot (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/search) databases. The nucleic acid and amino sequences of identified soybean NRAMP genes were downloaded from the Phytozome website. Soybean NRAMP genes were named according to phylogenetic relationships among the proteins. The chromosomal localization map and duplication of soybean NRAMP gene was determined by using ORTHOMCL (http://orthomcl.org/orthomcl/) and SVG softwares (http://search.cpan.org/~ronan/SVG-2.28/SVG/ Manual.pm). Protein molecular weights and theoretical pI values were computed using ProtParam (http://web.expasy. org/protparam/). Sequence identity among soybean NRAMP proteins was determined using BLASTP with each sequence queried against the other soybean NRAMP sequences in standalone BLAST downloaded from NCBI (https://blast.ncbi. nlm.nih.gov/Blast.cgi). Transmembrane helices in proteins were predicted using the TMHMM Server v. 2.0 (http://www. cbs.dtu.dk/services/TMHMM/). Predictions of subcellular localization for soybean NRAMP proteins were generated with ProtComp 9.0 (http://linux1.softberry.com/berry.phtml?group= programs&subgroup=proloc&topic=protcomppl). Multiple sequence alignment was performed with Clustal W and drawn in Genedoc, with the logo of consensus transport residues then generated by WebLogo 3 (http://weblogo.threeplusone. com/). Phylogenetic trees based on full length protein sequence alignments of NRAMPs from soybean and several other species were constructed by the neighbor-joining method with 1,000 bootstrap replicates in MEGA 6.0 (http://www.megasoftware. net/download\_form). Downloaded CDS and genomic sequences of soybean NRMAP genes were used to construct gene structures on the Gene Structure Display Server 2.0 (http://gsds.cbi.pku. edu.cn/index.php). PlantCARE (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/) was used to cis-element analysis in the 1,500 bp region upstream of the start codon for each NRAMP gene. ### Plant Materials and Treatments Soybean cv. Williams 82 was employed in this study. For tissue specific expression analysis of GmNRAMPs, soybean plants were cultured in hydroponics and harvested at a number of developmental stages for qRT-PCR assays. Specifically, soybean seeds were surface-sterilized in 10% H2O2, then, after germination for 1 week, seedlings were transplanted into fullstrength nutrient solution as previously described (Li et al., 2012) which contained 250 mM KH2PO4, 3,000 mM KNO3, 2,000 mM Ca(NO3)2, 250 mM MgSO4, 25 mM MgCl2, 12.5 mM H3BO3, 1 mM MnSO4, 1 mM ZnSO4, 0.25 mM CuSO4, 0.25 mM (NH4)6Mo7O24, and 25 mM Fe-Na-EDTA. The pH value of the nutrient solution was adjusted to 5.8, and nutrient solution was changed weekly. Seedlings were grown in a growth chamber with a 16 h light and 8 h dark cycle at 28◦C for 40 days before separately harvesting young leaves, older leaves, roots, stems, and flowers. At 55 days, young pods and seeds were also separately harvested. All tissue samples were stored at −80◦C for further RNA extraction and qRT-PCR analysis. To investigate possible responses of GmNRAMPs to nutrient deficiency, 10-day-old seedlings were exposed to low-nitrogen (LN), -phosphorus (LP), -potassium (LK), -iron (LFe), or -sulfur (LS) conditions for 14 days, in which time nutrient deficiency symptoms became evident. For the LN treatment, KNO<sup>3</sup> and Ca(NO3)<sup>2</sup> were replaced by K2SO<sup>4</sup> and CaCl2, respectively. For the LP treatment, KH2PO<sup>4</sup> was replaced by K2SO4. For LK, KNO3, and KH2PO<sup>4</sup> were replaced by Ca(NO3)<sup>2</sup> and NaH2PO4, respectively. For LFe, Fe-Na-EDTA was not added to the nutrient solution. In the LS treatment, MgSO<sup>4</sup> was replaced by MgCl2. Seedlings grown in full-strength nutrient solution were sampled as the control. Each treatment had four biological replicates. Leaves and roots were separately sampled for further RNA extraction and qRT-PCR analysis. To elucidate the probable functions of GmNRAMPs in response to divalent metal toxicity stresses, 10-day-old seedlings were treated with excess Fe (1,000 µM EDTA-Fe), Cu (200 µM CuSO4·5H2O), Cd (100 µM CdCl2), and Mn (200 µM MnSO4·H2O) treatments for 24 h. Each treatment had four biological replicates. Leaves and roots were separately sampled for further analysis. To further study the responses of GmNRAMPs to rhizobia inoculation, 7-day-old seedlings were inoculated with the effective rhizobial strain Bradyrhizobium sp. BXYD3 (Cheng et al., 2009), and then transplanted into low nitrogen (500 µM N added) nutrient solution. Each treatment had four biological replicates. Young leaves, stems, roots, and nodules were separately collected at 30 days after inoculation and then stored at –80◦C for RNA extraction and qRT-PCR analysis. ### RNA Extraction and qRT-PCR Analysis Total RNA was extracted from different soybean samples using RNAisoTM Plus reagent (Takara Bio, Otsu, Shiga, Japan) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNaseI (Invitrogen, Grand Island, NY, USA) to remove contaminating genomic DNA. The quality of total RNA was checked via spectrophotometry (TGem Plus, Tiangen, China). Then, first strand cDNA was synthesized using the PrimeScriptTM RT Master Mix (Takara, Tokyo, Japan) according to the manufacturer's protocol. For qRT-PCR analysis, the soybean housekeeping gene TefS1 (encoding the elongation factor EF-1a: X56856) was used as a reference gene, and specific primers for GmNRAMPs and TefS1 were designed with Primer-NCBI (https://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK\_LOC=BlastHome) as listed in Table S1. In addition, the specific primers for nutrient deficiency responsive genes were also designed and listed in Table S2. qRT-PCR reactions were carried out in a CFX connect Real-Time PCR Detection System (Bio-Rad, Hercules, USA) with SYBR <sup>R</sup> Premix Ex TaqTM II (TaKaRa, Tokyo, Japan). The PCR reaction volume was 20 µL in total, which included 2 µL diluted cDNA, 10 µL SYBR Premix Ex TaqTM reagent, 0.6 µL primers and 6.8 µL RNAfree water. PCR Reactions were performed under the following conditions: 95◦C for 1 min, followed by 40 cycles of 95◦C for 15 s, 60◦C for 15 s, and 72◦C for 30 s. The expression of NRAMP genes was calculated by the 2−1Cq and 2−11Cq methods (Livak and Schmittgen, 2001). ### Subcellular Localization and Predicted Protein Interaction Networks To determine the predicted subcellular localization of soybean NRAMP proteins, six GmNRAMP proteins were selected to generate subcellular localization constructs, then transient expression in Arabidopsis protoplasts, which were widely used in subcellular localization analysis of genes not only for soybean, but also for other plant species (Zhang et al., 2016; Chong et al., 2017; Chu et al., 2017; Péron et al., 2017; Xu et al., 2017). Specifically, the coding region of each GmNRAMP gene was amplified with gene-specific primers shown in Table S3. These CDS sequences were cloned into the pMDC43 vector to express GmNRAMP-GFP fusion proteins driven by the CaMV 35S promoter. The constructs of 35S::GmNRAMP-GFP and 35S:GFP (control) were separately transformed into Arabidopsis protoplasts. Arabidopsis protoplasts were isolated from the leaves of 4-week-old Arabidopsis plants and subsequently transformed according to previously published protocols (Yoo et al., 2007). After transfection using polyethylene glycol and incubation in a plate under weak light for 12–16 h, protoplasts were observed with an Olympus FV10-ASW laser scanning confocal microscope (Olympus, Japan). Corresponding markers used in coexpression experiment were selected according to predicted subcellular localizations of GmNRAMP proteins, which were also verified in rice protoplasts isolated from the stems of 10-day-old rice plants under dark culture conditions. The coexpression of two marker genes in rice protoplasts were performed same as the protocol for Arabidopsis protoplasts. To further investigate possible protein interactions involving GmNRAMPs, putative interaction networks were identified in the interaction section of the UniProt protein knowledgebase (http://www.uniprot.org/), with interactions originating from STRING 10.0 protein-protein interaction databases (http://www. string-db.org/). The default settings of association networks were applied in these analyses. ### Statistical Analysis All data were analyzed using Microsoft Excel 2010 (Microsoft Company, USA) for calculating mean and standard error. Comparisons of gene expression among genes and tissues using analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) for mean separation, as well as, in response to nutrient deficiencies using t-tests were performed in RStudio (RStudio, USA) using standard R packages (https://www.R-project.org). Resulting p-values from t-tests were corrected for false discovery in multiple hypotheses testing by manually calculating adjusted p (Q) values in Excel using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995). Comparisons of gene expression in response to metal toxicity and rhizobia inoculation were also performed using analysis of variance (ANOVA) in Excel. ### RESULTS ### Genome-Wide Identification and Bioinformatic Analysis of Soybean NRAMP Family Genes Thirteen putative GmNRAMP genes were identified in BLAST searches of the G. max cv. Williams 82 reference genome in the Phytozome database using arabidopsis, rice, and medicago NRAMP as query sequences. All identified GmNRAMPs were named based on phylogenetic relationships among soybean NRAMP family members (Figure 1A and Table 1), with the tree being comprised of two main branches (Figure 1A). The NRAMP family domain (PF01566) and 10–12 putative transmembrane domains (TMDs) were also found in each putative GmNRAMP protein (Table 1 and Figure S1). The CDS regions of putative GmNRAMP genes range in length from 1,521 to 1,767 bp and encode proteins with lengths of 506–588 amino acid residues, molecular weights of 55.44– 64.39 KDa, and pI values of 4.77–9.04 (Table 1). Gene structures were similar within each of the two main subfamilies as illustrated in Figure 1A. Subfamily I is comprised of 8 members, each with 4 exons and 3 introns, while the five Subfamily II GmNRAMPs each have 13–14 exons and 12– 13 introns. Among the Subfamily II proteins, GmNRAMP7, with a relatively short length, appears to be divergent from the other four members. It is also worth mentioning that GmNRAMP6a and GmNRAMP6b each contain 13 introns, with one intron located in the 5′ UTR (Figure 1A). Chromosome mapping showed that the 13 GmNRAMPs are distributed on 11 chromosomes. Chromosomes 6 and 7 each contained two NRAMPs, while chromosomes 1, 4, 5, 8, 11, 13, 15, 16, and 17 each contained one NRAMP (Figure 1B). From the phylogenetic tree of soybean NRAMP proteins, we noticed that all but GmNRAMP7 appeared in pairs, implying possible gene duplication occurred during evolution of NRAMP gene family, therefore, synteny analysis also performed to determine the potential gene duplication with soybean NRAMP family. As shown in Figure 1B, six pairs of soybean NRAMP genes were found to be located in duplicated blocks, suggesting the duplication event also happened in the soybean NRAMP gene family. In BLASTP analysis, all alignments included at least 73% of each GmNRAMP sequence, while 102 of the 169 alignments incorporated over 90% of the protein sequence (data not shown). Sequence identity in aligned regions ranged from 38 to 98% outside of self hits (Table 2), with the highest percentage of identity occurring between GmNRAMP3a and GmNRAMP3b which also fell very close to each other in the phylogenetic tree (Figure 1A). The lowest identity, on the other hand, occurred between GmNRAMP4b and GmNRAMP7 (Table 2). The reported consensus transport residues, GQSSTITGTYAGQY(/F)V(/I)MQGFLD(/E/N) were present in all identified soybean NRAMP sequences (Figure 2). Further phylogenetic investigation was conducted with the inclusion of NRAMPs from other plant species, bacteria, fungi and humans. As expected (Figure 3), NRAMPs from Deinococcus radiodurans (DraNRAMP), Staphylococcus capitis (ScaDMT), and Saccharomyces cerevisiae (ScSMF1, ScSMF2, and ScSMF3) were closely related and separated from higher plant and human NRAMPs (HsDMT1 and HsNRAMP1). The tested higher plant NRAMPs from soybean, arabidopsis, rice, medicago, barley, peanut, apple, and mustard, with one exception, fall into two large groups, subfamily I and subfamily II, with both monocots and dicots represented in each of these two groups. Arabidopsis carries four subfamily I members and two subfamily II members, while the corresponding subfamily I and II numbers were two and five for rice, and four and three from medicago. The 13 soybean NRAMP members sorted into eight subfamily I members and five subfamily II members, which matched the phylogenic and gene structure results described above. In addition, the tested NRAMP proteins from mustard clustered into subfamily I, while the NRAMP proteins from peanut, barley, and apple clustered into subfamily II (Figure 3). Interestingly, the subcellular localization predictions for GmNRAMPs indicate that subfamily I members localize to vacuoles, while subfamily II members localize to the plasma membrane (Table 1). To better understand the potential regulation of GmNRAMP genes, cis-element analysis was performed and shown in Table S4. A number of cisacting regulatory elements involved in light responsiveness, were frequently identified in soybean NRAMP genes promoter regions (Table S4). In addition, cis-regulatory elements in GmNRAMPs were also associated with various stress factors. Notably, the promoter regions of 9 NRAMP genes contained cis-elements related to defense and stress responsiveness (Table S4). Most NRAMP gene promoters contained at least one HSE element (heat stress responsiveness), which was followed in prevalence by MES (drought-inducible) and LTR (low temperature responsiveness) elements. Furthermore, several identified elements in GmNRAMP promoter regions have been reported to be involved in hormone responsiveness. Specifically, all GmNRAMPs except GmNRAMP6b were associated with the cis-acting element ABRE, which is involved in abscisic acid (ABA) responsiveness. Plus, the GmNRAMP2a and GmNRAMP4b promoter regions contained a series of elements responsive to nearly all types of hormone, including ABA, Methyl Jasmonate (MeJA), ethylene (ETH), gibberellin (GA), and auxin (IAA). These results indicate that GmNRAMP genes may be involved in complex regulatory networks and could be regulated by various environmental, developmental, and physiological factors. TABLE 1 \| Summary of NRAMP family genes in soybean. ### Tissue-Specific Expression of GmNRAMPs In order to investigate tissue-specific expression profiles of GmNRAMPs, qRT-PCR analysis was performed with seven soybean tissues, namely young leaves, older leaves, stems, roots, flowers, pods, and seeds. Using an ANOVA significance threshold of p = 0.05, expression varied among tissues for each of the 13 tested GmNRAMPs, as well as, among GmNRAMP genes within each tissue. Quantified expression levels for each GmNRAMP gene in each of the seven tissues are displayed in Figure 4, with those in the most highly expressed category according to Duncan's Multiple Range Test marked by an "a" for the tissues in which each gene was most highly expressed, and an "∗" for the most highly expressed GmNRAMPs in each tissue. Where GmNRAMP transcription was above the detection limit, expression levels varied by over 50-fold among tissues and GmNRAMP genes. Expression was low for GmNRAMPs 4b and 5b in all tissues, but was highest for both of these genes in flowers, as well as in pods for GmNRAMP4b. All GmNRAMPs were detected in each tissue, except seeds. The most highly expressed GmNRAMP in each tissue was GmNRAMPs 1a, 1b, 2b, 3b, or 7, with GmNRAMPs 1a, 1b, 2b, and 3b being notable for relatively high expression in multiple tissues. For leaves, relatively high expression was also observed for GmNRAMPs 2a, 3a, and 6a in young leaves, and for GmNRAMP 5a in old leaves. Higher expression was also observed for GmNRAMPs 6a and 6b in stems, and for GmNRAMP 5a in roots. In flowers and pods, appreciable expression was observed for all GmNRAMPs, except for 5a and 5b in pods. One notable result was that the most highly expressed GmNRAMPs typically belonged to subfamily II in all tissues, except in roots, where the subfamily I GmNRAMP 7 was the most highly expressed GmNRAMP. Taken together, structures, phylogenies (Figure 1) and expression patterns (Figure 4) demonstrate that structurally similar GmNRAMPs also exhibit similar expression patterns. ### Expression of GmNRAMPs in Response to Macronutrient Deficiency To evaluate potential responses of GmNRAMPs to macronutrient deficiencies, expression was assessed by qRT-PCR in soybean plants exposed to deficiencies of nitrogen (N), phosphorus (P), or potassium (K). GmNiR, which was repressed by N deficiency (Qin et al., 2012), together with GmPLDZ (a low P responsive gene) and GmHAK (a low K responsive gene), which had been demonstrated to respectively enhanced by P or K deficiency (Qin et al., 2012), were used to verify the nutrient deficiency treatments in this study (Figures S2A–C). Significant effects of macronutrient deficiencies were determined using FDR corrected t-tests for each GmNRAMP within each tissue for each nutrient treatment. As shown in Figure 5, the ratio of expression in deficient conditions relative to sufficient conditions significantly varied from constant expression for each GmNRAMP in one or more conditions and tissues. More specifically, N deficiency led to decreased expression of GmNRAMPs 1a, 2a, 2b, and 6a in leaves, and GmNRAMPs 5a, 6b, and 7 in roots, while GmNRAMP5a was up-regulated in leaves, and GmNRAMPs 3b and 6a were up-regulated in roots. Under P deficiency conditions, GmNRAMP expression was dramatically altered. Nine GmNRAMPs, 1a, 1b, 3b, 4a, 5a, 5b, 6a, 6b, and 7, were up-regulated by P deficiency in leaves. In roots, GmNRAMPs 3b, 5a, and 6a were up-regulated, while GmNRAMP7 was down-regulated. In comparison to N and P deficiency responses, K deficiency resulted primarily in decreased expression of GmNRAMP genes. GmNRAMPs 6a, 6b, and 7 were down-regulated in both leaves and roots, while GmNRAMP5b was down-regulated in roots, and, in leaves, GmNRAMPs 1a and 3b were down-regulated and GmNRAMPs 1b and 5a were upregulated. Among treatments and tissues, a few GmNRAMPs exhibited more notable responses. GmNRAMPs 5a, 6a, and 7 responded to all treatments, except for GmNRAMP7 in low N leaves and GmNRAMP5a in low K roots. Furthermore, GmNRAMP7 responses were in the negative direction, except in low P leaves, while GmNRAMP5a responses were in the positive direction, except in low N roots. Two GmNRAMPs, 3a and 4b, displayed considerable variation in relative expression among tissues and nutrient treatments (Figure 5), yet this variation was not significant due to the overall low level of expression for each of these genes (Figure 4). ### Expression of GmNRAMPs in Response to Iron or Sulfur Deficiency To further investigate potential roles for soybean NRAMP genes in nutrient homeostasis, GmNRAMPs were tested for alterations in expression in response to Fe or S deficiency. Two known marker genes, GmIRT (for low Fe) and GmSULTR1;2b (for low S) (Qin et al., 2012; Ding et al., 2016), were also applied to confirm the Fe or S deficiency in this study (Figures S2D,E). As shown in Figures 6, 9 GmNRAMP genes were significantly down-regulated by Fe deficiency. The abundance of GmNRAMP6a was downregulated in both Fe-deficient leaves and roots. GmNRAMPs 1a, 3a, 3b, 4a, 4b, and 6b were also down-regulated only in leaves, and GmNRAMPs 5a and 5b were also down-regulated only in roots. Four NRAMP genes responded to Fe starvation with enhanced expression levels. Among them, GmNRAMP2a and GmNRAMP2b exhibited increased expression both in leaves and roots, while GmNRAMP7 expression was enhanced in roots, and GmNRAMP1b was up-regulated in leaves. Furthermore, except for GmNRAMP2a and GmNRAMP5b, all soybean NRAMP genes also responded to S deficiency (Figure 6). Interestingly, responses to S deficiency were opposite of those observed for Fe deficiency, with the exception of three GmNRAMPs. Specifically, GmNRAMP5a was down-regulated in both Fe- and S-deficient roots, GmNRAMP1b was up-regulated in both Fe- and Sdeficient leaves, and GmNRAMP2a, responded to Fe, but not to S (Figure 6). ### Expression of GmNRAMPs in Response to Divalent Metal Toxicity Stresses In order to evaluate the probable functions of GmNRAMPs in responses to divalent metal toxicity stresses, expression of these genes was also assayed by qRT-PCR in soybean seedlings exposed to excess supply of Fe, Cu, Cd, or Mn. Due to low abundances of GmNRAMPs 4a, 4b, and 5b in leaves and roots under these treatments, only 10 NRAMP genes were analyzed in this experiment. In general, the expression of soybean NRAMP gene family members was most sensitive to Cd toxicity, followed by Cu toxicity. The expression of six GmNRAMP genes was greatly enhanced by excess Cd, with four of them were being up-regulated in both leaves and roots, and two of them were being enhanced in roots (Figure 7 and Table 3). While three NRAMP genes responded to Cd toxicity with reductions in expression levels (Figure 7 and Table 3). Under Cu toxicity stress, GmNRAMP5a was upregulated in both leaves and roots, and GmNRAMP1a was upregulated in roots, whereas the expressions of GmNRAMP6a and GmNRAMP2a were down-regulated in leaves and roots, respectively. In addition, two NRAMP genes, GmNRAMP2b FIGURE 2 \| Multiple alignment of soybean NRAMP family proteins. Multiple alignment was performed with Clustal W and the residues were colored using Genedoc software. Red lines underneath alignments indicate reported consensus transport residues. "\" above the sequence mean every ten amino acid residues. The logo of these residues was then generated in WebLogo 3 online. and GmNRAMP3a exhibited opposite trends in soybean leaves and roots under excess Cu stress. In another, the expression of soybean NRAMP genes seemed less influenced by excess Fe supply in comparison to the other toxicity treatments or to Fe deficiency, with only three genes, GmNRAMP2a, 2b, and 7 being suppressed by Fe toxicity (Figure 7* and Table 3). Finally, in the Mn toxicity treatment, expression was altered for only four NRAMP genes, with three being up-regulated, while one was down-regulated (Figure S3 and Table 3). ### Subcellular Localization of GmNRAMP Proteins To explore the subcellular localization of the GmNRAMP proteins, putative subcellular localizations were first identified by ProtComp analysis. As shown in Table 1, eight NRAMP proteins were predicted to localize to the vacuole, whereas the other five GmNRAMP proteins were predicted to target the plasma membrane. Subsequent to this computational analysis, subcellular localization for six selected GmNRAMPs proteins was empirically observed though transient expression of GFP: GmNRAMP fusions in Arabidopsis protoplasts containing the membrane marker OsMCA1 (Kurusu et al., 2012) or the tonoplast marker AtTPK1 (Voelker et al., 2006), with verification of these two specific localization makers conducted in co-transformed rice protoplasts (Figure S4). Microscopic observation revealed that the 35SGFP::GmNRAMP1a, 35SGFP::GmNRAMP2a, 35SGFP::GmNRAMP2b, and 35SGFP::GmNRAMP3a fusions were exclusively localized to the tonoplast, as evidenced by co-localization with the known tonoplast marker AtTPK1 (Figure 8A). The 35SGFP::GmNRAMP5a and 35SGFP::GmNRAMP7 fusions localized on the plasma membrane along with the membrane marker OsMCA1 (Figure 8B), and 35SGFP empty vector controls yielded whole cell fluorescence (Figure 8C). These results indicate that GmNRAMP proteins localize to different subcellular compartments. Furthermore, localization might be associated with specific biological functions in plant cells. ### Bioinformatic Analysis of Protein-Protein Interactions Involving GmNRAMPs To explore potential interactions among GmNRAMP members, protein-protein interaction analysis was conducted computationally in the Uniprot and STRING database. Predicted interacting proteins were nearly identical for 10 of the 13 GmNRAMP query proteins (network I in Figure S5A), including for GmNRAMP1a, GmNRAMP2a, and GmNRAMP4b to GmNRAMP7. All of the proteins predicted to interact with these 10 NRAMPs contain multicopper oxidase domains, and may be involved in the metabolism of ascorbate and aldarate (Table S5). In contrast to network I, GmNRAMP2b, GmNRAMP3a, and GmNRAMP3b were predicted to interact in another set of similar interaction networks as shown in Figure S5B. The putative networks for GmNRAMP3a, GmNRAMP3b, and GmNRAMP2b are very similar to each other, with seven identical interacting proteins labeled with a star in Figure S5B. These seven proteins include a Zn transporter, a vacuolar Fe transporter and a ferric reductase (Table S5). The interaction networks involving GmNRAMP3a and GmNRAMP3b share two proteins in common with network I as labeled with check marks, both of which contain multicopper oxidase domains. Meanwhile, GmNRMAP2b is predicted to interact with RBCS-1 (Ribulose bisphosphate carboxylase) and HDL56 (Transcription factor HEX, containing HOX, and HALZ domains). Interestingly, nodulin-21 and a nodulin-like protein were also found in the interaction networks of GmNRAMP2b, GmNRAMP3a, and GmNRAMP3b, suggesting potential functions for these GmNRAMPs in Fe or other metal ion transport in soybean nodules. ### Expression of GmNRAMPs in Soybean Nodules with Rhizobia Inoculation In order to determine whether GmNRAMP genes function in soybean nodules, the expression of GmNRAMPs were tested in different tissues of soybean 30 days after rhizobia inoculation. Due to low expression of GmNRAMP4a and GmNRAMP4b under low N conditions, only 11 GmNRAMP genes were evaluated in this experiment. As shown in Figure 9, expression of GmNRAMP1a, GmNRAMP1b, GmNRAMP6a, and GmNRAMP6b were lower in soybean nodules than in other tissues upon inoculation with rhizobia. Compared with non-inoculated soybean, expressions of GmNRAMPs 2a, 2b, 5a, 5b, and 6b were significantly reduced in soybean roots after inoculation with rhizobia (Figure 9), during which time expression in nodules ramped up considerably. Moreover, under rhizobial-inoculation conditions, expression of GmNRAMP2b, GmNRAMP3a, GmNRAMP3b, and GmNRAMP7also scaled up in soybean nodules, as indicated by the respective 21.66-, 13.96-, 11.96-, and 6.8-fold differences compared to expression in soybean roots (Figure 9). Higher expression of GmNRAMP2b, GmNRAMP3a, and GmNRAMP3b in nodules compared to other GmNRAMPs suggests that these genes might be important for the transport of Fe or other metal ions in soybean nodules. ## DISCUSSION NRAMP proteins are exist in a wide range of bacteria, animals and plants, to date, the NRAMP gene family has been reported in a number of plant species, but information on this family is limited for soybean, the most important cultivated legume. In this study, the soybean genome was comprehensively searched for NRAMP genes, which resulted in the identification of 13 putative GmNRAMP genes. Phylogenetic analysis clustered these 13 NRAMP proteins into two distinct subfamilies (Figure 1A). Interestingly, 12 of the 13 GmNRAMP proteins further clustered into six branches of paired proteins (Figure 1A and Table 2). These homologous pairs of GmNRAMPs might be the products of duplication events in soybean evolutionary history (Figure 1B). Altogether, the combination of homologous pairs and two subfamilies based on structural similarities guided the phylogenetically based naming of GmNAMPs employed herein. In Arabidopsis, the analysis of six NRAMP proteins has also revealed two subfamilies (Thomine et al., 2000; Mäser et al., 2001). Similarly to the current findings for soybean NRAMP genes, AtNRAMP2 through 5 in subfamily I have 2–3 introns, while AtNRAMP1 and AtNRAMP6 in the other subfamily have 10 and 12 introns (data not shown in paper). On the other hand, unlike AtNRAMP1 and AtNRAMP6, two soybean NRAMP genes in Subfamily II have an intron located on the 5′ UTR (Figure 1A). Previous studies have associated the presence of an intron within the 5′ UTR with enhanced RNA and protein accumulation (Rethmeier et al., 1997; Chung et al., 2006). In the current study, the two GmNRAMPs, GmNRAMP6a and GmNRAMP6b, which containing an intron within the 5′ UTR, were not among the most highly expressed genes in any of the tested tissues or conditions. Whether these 5′ UTR introns play roles in the regulation of GmNRAMP6a or GmNRAMP6b remains to be determined. Next, phylogenetic analysis of the 13 GmNRAMP proteins identified here, along with NRAMP protein sequences from other plant species (Figure 3), revealed that NRAMP proteins representing both subfamilies are common in both dicots and monocots. This suggests that the NRAMP family fulfills similar and basic functions in widely divergent plant species. Interestingly, soybean NRAMP proteins in the same subfamily were predicted and confirmed to have similar subcellular localizations (Figure 8 and Table 1). Such compartmentalization is likely related to specific functions, namely, uptake of metals on the plasma membrane, or release from vacuolar stores on the tonoplast as previously reported in Arabidopsis and rice. Specifically, AtNRAMP3 and AtNRAMP4 have been reported to be localized in Arabidopsis vacuoles, which is in consistent with roles in the release of metals from vacuolar stores (Lanquar et al., 2005). Meanwhile, OsNRAMP1, OsNRAMP3, and OsNRAMP5 have been identified as plasma membrane-localized proteins in rice participate in metal uptake (Takahashi et al., 2011; Sasaki et al., 2012; Yamaji et al., 2013). Phylogenetic analysis with these known NRAMP proteins implies potentially similar functions for soybean NRAMP proteins. NRAMP genes functions in metal ions uptake, especially Fe, are widely found in mice, humans and plants, particularly under Fe-deficiency conditions. Although the IRT/FRO system seems to be a major component of Fe-uptake system in the non-graminaceous plants, several previous researches revealed FIGURE 7 \| Expression of GmNRAMPs in response to different metal toxicity stresses. Ten-day-old seedlings were treated with excess Fe (1,000 µM EDTA-Fe), Cu (200 µM CuSO4·5H2O), and Cd (100 µM CdCl2) treatments for 24 h. Each bar is the mean of four biological replicates with standard error. "\" indicates effects of toxicity treatments relative to controls in one-way analysis of variance, \P < 0.05, ns: not significant. TABLE 3 \| Summary of the response of soybean NRAMP genes to different divalent metal toxicities. Significant differences between treatment and control are marked as arrows. "↑" stands for up-regulated expression in response to the treatment, while "↓"stands for down-regulated expression in response to the treatment. that other genes could be involved in this process, such as NRAMP genes. In Arabidopsis, AtNRAMP1 can complement the Fe uptake mutant of yeast, and appears to be expressed preferentially in Fe-deficient roots, indicating its role in Fe uptake and transport (Curie et al., 2000). In soybean, 13 GmNRAMP genes were significantly affected by Fe deficiency in leaves or roots of soybean (Figure 6), indicating that GmNRAMP genes also are involved in Fe nutrition. For root-expressed GmNRAMP genes, GmNRAMP7 was remarkably up-regulated by Fe deficiency in roots (Figure 6), combining with its plasma membrane localization (Figure 8), implying that GmNRAMP7 might participate in the acquisition of Fe on the plasma membrane in root cell, perhaps also coupling with IRT and FRO as integral part of plant root cell Fe uptake machinery under Fe starvation stress. However, this hypothesis would be further investigated by the tissue and cell specific localization of GmNRAMP7 by GUS staining and GFP fluorescence in future trails. Besides, previous study showed that overexpression of AtNRAMP1 leads to an increased resistance to toxic Fe level (Curie et al., 2000), indicating it may participates in Fe remobilization under Fe deficiency, despite this function was not consistent with its plasma membrane localization. However, it is uncertain that this kinds of gene always going to be present in cell membranes? It's interesting to speculate that if AtNRAMP1 or GmNRAMP7 was not always present in cell membrane, whether it might performed potential functions in Fe or other metals transport in root cell, for example, remobilization of Fe stored in organelles under Fe-deficient conditions as MxNRAMP1 which mainly exists in the plasma membrane and vesicles (Pan et al., 2015), or regulate homeostasis of free ions which might induced by Fe starvation, mediate sequestration of free ions into a cellular compartment, such as plastid or vacuole, all of these speculations also need further study. Beyond functioning in Fe transport, NRAMP genes have been also demonstrated to perform wide ranging transport activities for divalent transition metals, including Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2<sup>+</sup> (Illing et al., 2012). Recently, several studies have reported the substantial role of NRAMP family members in Mn uptake. As shown in Figure 3, four NRAMP proteins, GmNRAMP5a, GmNRAMP5b, GmNRAMP6a, and GmNRAMP6b cluster together into a small phylogenetic branch with AtNRAMP1, a plasma membrane localized high-affinity Mn transporter (Cailliatte et al., 2010). This small branch is also closely related to OsNRAMP3, which is a known Mn transporter that is involved in shoot Mn distribution, and is constitutively expressed in nodes, stems and panicles, where it mediates adaptation of rice to a wide change of external Mn conditions (Yamaji et al., 2013; Yang et al., 2014). Interestingly, one soybean NRAMP protein, GmNRAMP5a, clustered in the phylogenetic tree with AtNRAMP1 and OsNRAMP3 (Figure 3), and was also affected by Mn as indicated by the significant increase in its expression in response to Mn toxicity (Figure S3 and Table 3), suggesting it is probably important for Mn homeostasis in soybean. In addition, NRAMP genes also seem to affect the intracellular remobilization of divalent toxic heavy metals, such as Cd (Cailliatte et al., 2009), which might contribute to increase plant tolerance to heavy metal toxicity. In this study, the responses of GmNRAMP genes to different divalent metal toxicities were also investigated (Figure 7 and Table 3). As expected, the remarkable changes in expression were observed for most GmNRAMPs under excess Cd treatment, suggesting that these genes might contribute to Cd tolerance in soybean plants under Cd toxicity stress. Interestingly, among 13 soybean NRAMP genes, the expressions of GmNRAMP1a, GmNRAMP3a, GmNRAMP5a in roots were both dramatically enhanced by Cu and Cd toxicities (Figure 7 and Table 3), furthermore, the expression of GmNRAMP5a in roots were significantly increased by Cu, Cd, and Mn toxicities (Figure 7, Figure S3 and Table 3). In consequence, we propose that soybean NRAMP genes widely participated in Fe, Mn, Cu, and Cd transport, might be involved in the uptake and homeostasis regulation of these metal ions. Moreover, considering the fact that macronutrient deficiencies often exist in field conditions, the responses of soybean NRAMP genes to N, P, and K deficiencies were also evaluated in this study. Most GmNRAMP responses to N deficiency involved down regulation, while, in contrast, a majority of GmNRAMP genes were remarkably enhanced by P deficiency (Figure 5). This enhancement of NRAMP gene expression under low P conditions is similar to patterns observed for soybean Fe-S assembly genes (Qin et al., 2015). A considerable amount of research has outlined interactions between P and Fe response pathways in plants. Phosphorus deficiency can increase carbon flux through glycolysis for the synthesis of organic acids, change lipid metabolism, and affect the abundance of genes involved in Fe and Zn metabolism (Wasaki et al., 2003; Zheng et al., 2009). Notably, increased Fe concentrations are often observed in P-deficient plants (Misson et al., 2005; Hirsch et al., 2006). The correlated Fe and P responses described in the current and previous studies indicate the existence of linkages between P and Fe metabolism through one or more pathways, including those involved with transport, homeostasis and accumulation. In previous work, P deficiency affected Fe storage, as indicated by the accumulation of Fe associated with ferritin in chloroplasts (Hirsch et al., 2006). More recently, cross-talk between P and Fe homeostasis pathways has been demonstrated in Arabidopsis, where the ferritin gene, AtFer1, is regulated by the phosphate starvation response transcription factor AtPHR1 (Bournier et al., 2013). In contrast, P deficiency does not appear to affect Arabidopsis genes encoding strategy I Fe-uptake system proteins, such as IRT1 (Hirsch et al., 2006). Whether the expression of other Fe uptake transporters is triggered in P-deficient Arabidopsis remains unknown. In this study, significant changes in the expression of GmNRAMP genes in P-deficient soybean plants reinforces the idea that plant P and Fe response pathways are linked and interact, and further imply that low P impacts Fe uptake and homeostasis through the activities of GmNRAMP transport systems. However, comprehensive elucidation of linkages between P and Fe metabolic pathways and the underlying mechanisms requires further investigation. Interactions between P and Fe pathways in plants has also been observed in jasmonate ZIM domain (JAZ) genes, which act as transcriptional repressors of jasmonateresponsive genes, as indicated by highly altered expression of such genes in rice and chickpea experiencing mineral nutrient deficiency (Singh et al., 2015). What's more, observations of the involvement of JAZ genes in nutrient deficiency responses contribute to further understanding of the roles jasmonates play in regulating nutrient deficiency response adaptations. It is noteworthy that several cis-acting elements involved in hormone-responsiveness were found in the putative promoter regions of soybean NRAMP genes (Table S4), but the regulatory mechanisms remain unclear. Whether NRAMP involvement in the uptake or homeostasis of metal ions is related to hormoneregulated morphological and physiological responses to nutrient and toxin status requires further study. One final interaction relevant here is the coordination of Fe with S in Fe-S proteins, which are the biggest Fe sink in plants. Unsurprisingly, therefore, interplay between Fe and S has been noted in recent years (Zuchi et al., 2015). Interestingly, most soybean NRAMP genes displayed contrasting responses to Fe and S deficiencies (Figure 6). Since Fe-S clusters are significant Fe sinks within cells, the deprivation of Fe or S will depress the synthesis of Fe-S clusters. Changes in GmNRAMP expression in response to Fe- or S-deficiency suggest links between these two stress responses. More definitive demonstrations of whether NRAMP genes are involved in coordinated regulation of Fe and S homeostasis, as well as, the synthesis of Fe-S clusters requires further investigation. It is worth mentioned that several soybean NRAMP proteins were not only found to interact with metal ion transporters, such as the Zn/Fe transporter, but several were also observed in interactions with nodulin and nodulin-like protein (Figure S5 and Table S5). The high abundance of GmNRAMP2b, GmNRAMP3a, and GmNRAMP3b in soybean nodules confirmed a predicted interaction network (Figure S5B) and suggested possible functions for these GmNRAMP genes in soybean-rhizobia symbiosis. Besides these three NRAMP genes, GmNRAMP2a and GmNRMAP7 were also detected in nodules at relatively high expression levels (Figure 9). However, these two proteins were not predicted to interact with nodulationrelated proteins. The expression patterns of GmNRAMP2a in various soybean tissues subsequent to rhizobia inoculation did not correspond to those of its homolog GmNRAMP2b, which might be due to the evolutionary divergence. As shown in Figure 3, the nodule-expressed subfamily II gene GmNRAMP7 has diverged from the other four nodule-expressed NRAMP genes. GmNRAMP7 is the only soybean NRAMP protein in a small branch with several monocot NRAMPs (including OsNRAMP1, OsNRAMP5, and HvNRAMP5), which are mainly expressed in roots (Sasaki et al., 2012; Wu et al., 2016). This is consistent with the tissue-specific expression of GmNRAMP7 (Figure 4). It also clusters with AhNRAMP1, a Fe transporter proven to be involved in Fe acquisition (Xiong et al., 2012). Most notably, GmNRAMP7 also clusters closely with MtNRAMP1, which has been reported to be most highly expressed in roots and nodules, as well as, the main transporter responsible for Fe uptake in nodule cells (Tejada-Jiménez et al., 2015). Taken together, the results herein imply that GmNRAMP7 might function in Fe uptake during symbiotic nitrogen fixation. Finally, GmNRAMP7 is predicted to be localized to the plasma membrane, while the four other nodule-expressed NRAMP genes were predicted to localize on the tonoplast. Any specific roles for these genes in the regulation of metal ions homeostasis requires further study. ### CONCLUSION In the present study, 13 NRAMP genes were identified from the soybean genome and, subsequently, named according to the phylogenetic relationships inferred among them. Expression profiles of GmNRAMP genes varied among soybean tissues and in response to a series of nutrient stresses, which suggests that GmNRAMPs perform a range of functions in specific tissues throughout growth and development. Furthermore, this gene family also likely participates in crosstalk among different nutrient stress pathways. Then the subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of soybean NRMAP proteins. Taken together, the results reported here comprise a systematic genome-wide analysis of the soybean NRAMP gene family. These results supply basic and important information for understanding the putative functions of NRAMP genes in soybean. Moreover, protein-protein interaction network and qRT-PCR analysis in rhizobia-infected soybean further revealed that 3 NRAMP proteins putatively interact with nodulin-like proteins and are markedly up-regulated in soybean nodules. These results suggest potential functions for a subset of GmNRAMP proteins in the uptake of Fe or other metals, as well as, regulation of homeostasis in soybean nodules. Overall, this study provides valuable information for further functional studies on the biological roles of NRAMP genes in soybean. Plus, this report provides a basis for further understanding crosstalk between different nutrient response pathways in plants. ### AUTHOR CONTRIBUTIONS LQ and XL conceived the study, analyzed the data, and drafted the manuscript. LQ, XH, and LX cultivated the soybean materials and collected the soybean samples. PH, LC, and YL extracted ### REFERENCES RNA and performed the qRT-PCR experiments. LC designed the vectors for subcellular localization. TW conducted the statistical analyses of raw data. HL and TW revised the manuscript. All authors read and approved the final manuscript. ### ACKNOWLEDGMENTS This research was financially supported by National Natural Science Foundation of China (Grant No. 31301833), Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2013-OCRI) and Excellent Young Scientist Fund (1610172015004) of Chinese Academy of Agricultural Sciences. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 01436/full#supplementary-material in Arabidopsis with homology to Nramp genes. Proc. Natl. Acad. Sci. U.S.A. 97, 4991–4996. doi: 10.1073/pnas.97.9.4991 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Qin, Han, Chen, Walk, Li, Hu, Xie, Liao and Liao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Harnessing Finger Millet to Combat Calcium Deficiency in Humans: Challenges and Prospects Swati Puranik <sup>1</sup> , Jason Kam<sup>1</sup> , Pranav P. Sahu<sup>1</sup> , Rama Yadav <sup>1</sup> , Rakesh K. Srivastava<sup>2</sup> , Henry Ojulong<sup>3</sup> and Rattan Yadav <sup>1</sup> \* 1 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom, 2 International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India, <sup>3</sup> International Crops Research Institute for the Semi-Arid Tropics, Nairobi, Kenya Humans require more than 20 mineral elements for healthy body function. Calcium (Ca), one of the essential macromineral, is required in relatively large quantities in the diet for maintaining a sound overall health. Young children, pregnant and nursing women in marginalized and poorest regions of the world, are at highest risk of Ca malnutrition. Elderly population is another group of people most commonly affected by Ca deficiency mainly in the form of osteoporosis and osteopenia. Improved dietary intake of Ca may be the most cost-effective way to meet such deficiencies. Finger millet [Eleusine coracana (L.) Gaertn.], a crop with inherently higher Ca content in its grain, is an excellent candidate for understanding genetic mechanisms associated with Ca accumulation in grain crops. Such knowledge will also contribute toward increasing Ca contents in other staple crops consumed on daily basis using plant-breeding (also known as biofortification) methods. However, developing Ca-biofortified finger millet to reach nutritional acceptability faces various challenges. These include identifying and translating the high grain Ca content to an adequately bioavailable form so as to have a positive impact on Ca malnutrition. In this review, we assess some recent advancements and challenges for enrichment of its Ca value and present possible inter-disciplinary prospects for advancing the actual impact of Ca-biofortified finger millet. Keywords: finger millet, calcium, osteoporosis, bioavailability, food processing, biofortification, genetic improvement, plant breeding ### IMPORTANCE OF CALCIUM IN HUMAN DIET Calcium (Ca) is the fifth most abundant element present in the human body, accounting for up to 1.9% of the body weight in adults (Nordin, 1976). Its main functions are to provide rigidity and structure, mediating vascular and muscular contractions or dilations and nerve signal transmission (Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, 1997; Nordin, 1997). Ca may also serve in the protective role against various types of cancer viz. colorectal (Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, 2011), ovarian (Goodman et al., 2002), breast (Lin et al., 2007), and prostate (Gao et al., 2005). Although not supported by clinical trials, observational studies have associated higher Ca intakes to lower body weight and reduced adiposity, which may be due to lower intracellular Ca in fat cells leading to a higher fat breakdown (Parikh and Yanovski, 2003). Thus, it may reduce the risk of cardiovascular diseases by lowering intestinal lipid absorption, promoting lipid excretion and decreasing cholesterol concentrations in the blood #### Edited by: Raul Antonio Sperotto, Centro Universitário UNIVATES, Brazil #### Reviewed by: Sukhwinder Singh, International Maize and Wheat Improvement Center, Mexico Prashant Vikram, International Maize and Wheat Improvement Center, Mexico Charu Lata, National Botanical Research Institute (CSIR), India ### \Correspondence: Rattan Yadav [email protected] #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science > Received: 17 May 2017 Accepted: 12 July 2017 Published: 26 July 2017 #### Citation: Puranik S, Kam J, Sahu PP, Yadav R, Srivastava RK, Ojulong H and Yadav R (2017) Harnessing Finger Millet to Combat Calcium Deficiency in Humans: Challenges and Prospects. Front. Plant Sci. 8:1311. doi: 10.3389/fpls.2017.01311 369* (Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, 2011). Ca is also known to be important for those who have diabetes (Levy et al., 1994; Vestergaard, 2006; Pittas et al., 2007), particularly type 1 diabetics, who in general have lower bone mineral density than healthy subjects (Ma et al., 2012; Oei et al., 2013). Given its importance, authorities like the Food and Agriculture Organization (FAO) of the United Nations have set up a recommended daily intake (RDI) of Ca based on age, life stage and gender (Food Agricultural Organization of the United Nations, 2002). During the phases of active growth, Ca equilibrium in the body maintains a stable bone mass. Therefore, FAO recommends that children of 1–3 years consume 500 mg Ca/day, 4–6 years consume 600 mg Ca/day and 7–9 years consume 700 mg Ca/day, which should be increased to 1,300 mg/day during 10–18 years (Food Agricultural Organization of the United Nations, 2002). About 1,000 mg Ca/day is recommended between the ages of 19–65 years in males. The organization also advocates that women should take 1,000 mg Ca each day from 19 years onwards until menopause raising it to 1,200 mg during the last trimester of pregnancy, and to 1,300 mg from 65 years and above (Food Agricultural Organization of the United Nations, 2002). However, after 50 years in men and in menopausal women, the onset of bone decalcification and demineralization leads to reductions in bone mass causing the disease osteoporosis (Michaelsson et al., 2005). According to International Osteoporosis Foundation, it is a significant problem both in the developed as well as in developing nations (http:// www.iofbonehealth.org/facts-statistics). The World Health Organization (WHO) has declared osteoporosis as the next main public healthcare concern globally, after cardiovascular diseases (CVDs), inflicting almost 75 million people in Europe, the United States of America and Japan alone (Consensus Development Statement, 1997; Haldipur, 2003). With a growing elderly population, osteoporosis is threatening to become a major global economic burden for the already stretched healthcare system across the globe. By 2050, the worldwide cost of treating osteoporosis is forecasted to USD 131.5 billion (Lindsay et al., 2001). Thus, in order to prevent Ca deficiency at this age, it is necessary to sustain the recommended daily intake (RDI) of 1,300 mg/day (especially after 65 years; Food Agricultural Organization of the United Nations, 2002) to ensure maximal bone mass during the developmental stages and to have reduced bone mass loss during old age (Michaelsson, 2009). Despite the importance of adequate Ca intake for human health and wellbeing, the WHO estimates that low dietary intake of Ca is common across the world (WHO, 2006). On a global scale, it presents a large risk which gets graver in underdeveloped regions of the world. However, the absence of reliable and practical indicators in these areas provides insufficient data, which is a challenge for resolving the actual global status for the prevalence of Ca deficiency. It has recently been determined (mainly based on food supply) that 3.5 billion people were at the risk of Ca deficiency in 2011, with approximately 90% of the affected individuals in Africa and Asia (Kumssa et al., 2015). Most of these regions have an agriculture-based economy and large segments of these populations are typically dependent on what they grow and produce for their Ca need. In such situations, staple crops that can offer adequate Ca requirements, especially for people of low income groups in these countries, are highly recommended. One such Ca-rich, traditional and locally well-adapted crop is finger millet. As opposed to nutritionally deficient cereals, such as rice, its regular consumption has a vast potential to curb the incidences of Ca deficiency. Finger millet also possesses many other health-benefitting traits. For example, it has been highlighted that as a model nutraceutical crop, finger millet can provide excellent solutions to food and health security issues (Kumar et al., 2016a). Being a stress resilient crop requiring minimal inputs for growth it is especially suited for sustainable agriculture (Gupta et al., 2017). Despite this, it has received very little scientific attention, relative to other crops, such as rice, wheat and maize. Some very recent reviews have provided a comprehensive account of molecular mechanisms that may be involved in calcium nutrition in finger millet and how various approaches, including molecular breeding, functional genomics and transgenic technology can elevate Ca accumulation in its grains (Ganapathy, 2017; Sharma et al., 2017). However, beyond this, in order to advance biofortification of finger millet, challenges associated with its impact on human health also needs to be critically evaluated. With this as focus, we have not only attempted to highlight its potential but also the prospects of advancing the bioaccessibility of grain Ca content even further. ### STRATEGIES TO PREVENT CALCIUM DEFICIENCY Calcium (Ca) deficiency is almost physically undetectable and difficult to diagnose during preliminary stages (Wang et al., 2013), however, if detected, it is usually easy to treat by increasing dietary Ca intake or absorption. Currently, there are many strategies that can be undertaken to manage the problem of Ca deficiency. These mainly include diversification of diet, food fortification, external supplementation and crop biofortification. Dietary changes by including foods that are naturally rich in Ca, like dairy products, seems to be the most efficient way to prevent Ca deficiency. However, it is not easy to persuade people to go for diversified diets and certain foods cannot be included in the diets. For example, 65% of the world's population is lactoseintolerant and therefore they cannot rely on dairy products for their Ca requirement. Incidentally, most of these lactoseintolerant people live in Asian and African regions (Curry, 2013), which are predominantly dependent on agriculturebased economies. Additionally, due to resource constrain, they may not be able to afford livestock or may solely raise cattle for supplementing income and deny themselves of milk and milk products. Therefore, many communities in these regions, including vegetarians and vegans, need alternative sources to meet their Ca needs. Another strategy can be an industry-based fortification of foods consumed by target groups. For example, policy decisions in the western world have supported fortification of breakfast cereals, fruit juices, flours and sports drinks with Ca. However, fortification may alter flavor, bioavailability, shelf life or safety of the product, thus making it unpalatable to the consumers. Further, consumers in under-developed nations, with limited education and economic resources, often do not always have access to such fortified foods. Supplementation is an alternative in which Ca tablets are used to prevent Ca deficiency. Although more accessible to people, this strategy suffers from major drawbacks, mostly associated with their side-effects (Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, 2011). The most common content of supplementation pills are the inorganic forms: calcium carbonate and calcium citrate. Large amounts of Ca supplements cause excessive Ca accumulation in vascular and soft tissues like arteries or organs, such as kidneys which can lead to heart attack or kidney stones, respectively (Bolland et al., 2010; Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, 2011). Some studies have also linked supplement intake with higher risk of breast cancer, prostate cancer and cardiovascular diseases (Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, 2011). Further, taking Ca supplements with meals may also reduce absorption of other minerals like zinc and iron (Cook et al., 1991). Therefore, even though this strategy can potentially reach many people, the associated medical problems make it a non-viable long-term option. To combat Ca deficiency for a wider and more sustainable impact, alternative solutions that are cost-effective and can easily be adopted especially in the developing countries settings need to be pursued. Genetic biofortification is one such very powerful plant breeding or transgenics-based strategy which can combat the global challenge of micronutrient deficiency (www.copenhagenconsensus.com). As it involves the already established agricultural systems to grow, breed and distribute nutrient-dense staple crops, it can be the most economically and socially feasible approach to integrate nutrition into the diets of the impoverished. Biofortified staple crops have been quantified to have high potential benefits and cost effectiveness as their cost per disability-adjusted life years (DALYs) saved is less than the national per capita income, when compared to other interventions, such as fortification and supplementation (Stein et al., 2005; Asare-Marfo et al., 2014). Over the last decade several projects, such as those initiated by Harvest Plus, have released nutritionally enhanced rice and wheat varieties (Bouis et al., 2011). Recently, efforts have also commenced for biofortification breeding of small cereals, such as millets. Miles ahead of widely consumed rice and wheat, this group of hardy cereals contribute up to 75% of the total calorie intake for poorest of the poor (O'Kennedy et al., 2006). Therefore, millets hold stronger promise to provide economic, food and nutritional security to people surviving on marginal and resource-poor soils (Muthamilarasan et al., 2016). In this respect, pearl millet has become a target for biofortification of iron and zinc (Manwaring et al., 2016) and has entered the spotlight in combating diabetes (Kam et al., 2016). Similarly, finger millet is also gaining popularity as an ideal target crop for Ca biofortification. ### WHY USE FINGER MILLET AS A MODEL FOR CALCIUM BIOFORTIFICATION? Finger millet possesses all the quantitative and qualitative traits to serve as a model for Ca biofortification. It stands out as the richest source of Ca among all the cereals (Table 1). It has three times more Ca than milk and 10-fold higher Ca than brown rice, wheat or maize (Kumar et al., 2016a). Besides Ca, finger millet is also very rich source of iron, amino acids like methionine, slowly digestible starch and phytochemicals like polyphenols. It is a gluten-free, low fat cereal which is non-allergic and easily digestible. For these characteristics, it is often termed as a "super cereal" (Kumar et al., 2016a). Apart from its nutritional attributes, finger millet has excellent environmental sustainability credentials. It can easily withstand harsh climatic conditions, low soil fertility, requires very little inputs with a short growing season (Kumar et al., 2016a). It can reach the yield potential of up to 10 tons/ha under optimum irrigated conditions (Padulosi et al., 2015). It has excellent storage quality traits and can be valuable in areas where farmers suffer losses due to dearth of post-harvest management. Therefore, integration of a naturally Ca-rich crop like finger millet in global biofortification programs can be a good starting point to alleviate Ca malnutrition (Sharma et al., 2017). Given that women share a significantly higher proportion of osteoporotic morbidity ("Facts and Statistics," International Osteoporosis Foundation, http://www.iofbonehealth.org/factsstatistics), regular consumption of finger millet during and after pregnancy as well as lactation can provide significant benefits to maternal and child bone health. Another advantage of Ca-enriched finger millet over expensive commercially fortified foods is its affordability to these malnourished areas. For lowincome households which mostly subsist on starchy and bulky foods like rice and cassava for their calorie requirements (https:// www.devex.com/news/better-crops-for-better-nutrition-86583), finger millet ensures a pragmatic solution that no family member (especially women and children) suffers from Ca deficiency. ### PROSPECTING BIOAVAILABILITY OF CALCIUM FROM BIOFORTIFIED FINGER MILLET TO ACHIEVE ADEQUATE INTAKES Determining the efficacy and biological impact of Ca-biofortified finger millet on better nutrition and improved health is very challenging. It depends mainly on two processes: bioaccessibility and bioavailability of Ca in the seeds. Bioaccessibility is a measure of the nutrient fraction available for absorption after its release from food matrix in the gastrointestinal tract. On the other hand, bioavailability is a utilization-based definition, where the ingested, digested and absorbed nutrient reaches the systemic circulation and exerts a positive effect on health (Carbonell-Capella et al., 2014). Solubility, dialysability and gastrointestinal model are generally used as in vitro methods for measuring Ca bioaccessibility. On the other hand, evaluation of Ca bioavailability is ideally evaluated through in vivo human studies. However, considering the complexity of large-scale human trials and ethico-legal procedures, Caco-2 cell culture, which behave like human intestinal cells, can provide an alternative analysis. It is well known that only <30% of the consumed Ca is effectively absorbed (Heaney, 2006). Interestingly, using the in vitro bioaccessibility methods, uncooked finger millet has been found to have 36.6% soluble and 28% dialyzable and bioavailable Ca (Amalraj and Pius, 2015). This is higher than rice (30.4% soluble Ca; 24.7% dialyzable Ca), sorghum (31.9% soluble Ca; 26.0% dialyzable Ca) and maize (25.4% dialyzable Ca). Therefore, finger millet in itself is an effective source of bioavailable Ca than many other staple cereals and its improvement through biofortification is an effective strategy that can relegate Ca deficiency. However, the fate of actual Ca bioavailability from finger millet relies on and is challenged by several other factors. These include intrinsic grain property (solubility, interaction with other constituents of the food matrix) and extrinsic factors (condition of the host, food processing and storage). Therefore, a better understanding of how these factors influence and impact Ca efficacy becomes essential before the biofortified finger millet can carve a path in the farmer and consumer markets. ### Grain's Intrinsic Factors That Impact Ca Bioavailability in Finger Millet Many plant-based Ca sources have limited accessibility of Ca for absorption due to the formation of insoluble complexes. Phytate and oxalate are two such bioavailability limiters that can impede Ca absorption as they exhibit a strong negative correlation with Ca bioaccessibility (Kamchan et al., 2004; Gibson et al., 2010; Krishnan et al., 2012). Several studies in legumes and cruciferous vegetables have reported that high in vitro Ca solubility and dialysability corresponds to low levels of phytate, oxalate and dietary fiber (Lucarini et al., 1999; Kamchan et al., 2004). In cereals, phytate and oxalate were shown to account for 7 and 15–20% inhibition of Ca bioavailability, respectively (Amalraj and Anitha Pius, 2015). In wheat and barley, phytate, but not fiber, has been proclaimed as having the major inhibitory effect on Ca absorption (Kennefick and Cashman, 2000). Phenolic compounds like tannins reduce the bioavailability of minerals by forming insoluble complexes with divalent metal ions (Rao and Prabavathi, 1982). An in vivo digestibility trial on birds fed on low (1%), medium (2%), and high (3%) tannin sorghum diets showed that as compared to control, the Ca absorption reduced by 1.22, 1.67 and 2.22 fold, respectively (Mahmood et al., 2014). Likewise, finger millet also contains these antinutrients that negatively affect grain palatability and can be a constraint to its Ca bioaccessibility. There is a wide range of phytate and oxalate content in finger millet based on the genotypes. The phytate content in finger millet ranges from 679 mg/100 g to 1,419.4 mg/100 g (Antony and Chandra, 1999; Makokha et al., 2002). The grains have been found to contain higher phytate content (783.5 mg/100 g) than rice (289.9 mg/100 g), pearl millet (518.5 mg/100 g) and sorghum (571.1 mg/100 g) but lower than wheat (792.1 mg/100 g) and maize (851.5 mg/100 g) (Amalraj and Pius, 2015). Similarly, finger millet grains have been reported to contain oxalic acid to the extent of 45.7 mg/100 g (Rachic and Peters, 1977). Out of the total oxalate fraction present in the food matrix, soluble oxalate has the ability to bind Ca and reduce its absorption. In a recent study, it was found that finger millet has higher total oxalate content (11.3 mg/100 g) than other cereals (except pearl millet; 20.0 mg/100 g) but had the lowest percentage of soluble oxalate (45.9%) among other cereals (Amalraj and Pius, 2015). Even though the phytate and total oxalate content of finger millet are higher than many other cereals, it still contains more bioavailable Ca percentage (28%) than rice (24.7%), maize (25.4%), and sorghum (26%) (Amalraj and Pius, 2015). Finger millet grains also have a wide range of total phenolics and tannins content (Devi et al., 2014). Tannin content estimation has revealed that the African varieties of finger millet have about three times more tannin percent than the Indian varieties (Ramachandra et al., 1977). Finger millet has been shown to contain up to 264.1 mg/100 g tannin (Amalraj and Pius, 2015). This is much higher than maize (25.5 mg/100 g) and rice (14.3 mg/100 g) but lower than pearl millet (275.8 mg/100 g) and wheat (287.3 mg/100 g) (Amalraj and Pius, 2015). Despite the knowledge of the extent of varietal variations in tannin content of finger millet, their direct effect on inhibition of Ca absorption and bioavailability has yet not been investigated. Calcium absorption may also be affected by the non-digestible oligosaccharides and dietary fibers in the food matrix. These compounds reduce the activity of digestive enzymes and slow down the digestion process. Dietary fibers were found to be significantly associated to Ca bioavailability in commercially available rice flakes (Suma et al., 2007). The indigestible starch component (resistant starch) has been found to increase Ca absorption in rats, probably by enhancing its solubility (Schulz et al., 1993). Finger millet is also a great source of resistant starch (Devi et al., 2014). It also has the highest total, soluble and insoluble dietary fiber when compared with wheat, rice, maize, sorghum and pearl millet (Amalraj and Pius, 2015). The role of such factors present in finger millet and other grain crops is generally considered positive in Ca absorption but the magnitude of their stimulatory effect requires further validation by in vitro or in vivo methods. For enhanced Ca bioavailability from finger millet, grain Ca content needs to be improved with a concomitant but conscious effort for the reduction of antinutrient compounds. This is because these compounds play a vital role in plant development and survival. For example, finger millet tannins are effective in reducing pre- and post-harvest losses as they provide protection against molds, insects and other abiotic stress (Gull et al., 2014). Similarly, phytic acid acts as the main phosphorus store for the seeds (Singh and Raghuvanshi, 2012). These compounds have also called attention due to their nutraceutical value and protective effects against many chronic diseases (Kumar et al., 2016a). Thus, their importance can never be completely disregarded. Engineering their content to become a non-limiting factor in Ca absorption from finger millet must be done in a way that does not negatively affect crop performance. A justified way to accomplish this is by employing efficient and suitable grain processing techniques. ### Influence of Grain Processing on Ca Bioavailability Processing techniques of the grain can affect the total mineral content and factors associated with their bioaccessibility. Finger millet is a very versatile cereal and can be processed and utilized in numerous ways while retaining the available Ca. Popping by high temperature and short time (HTST) treatment was found to have no effects on the total Ca content in finger millet but lowered the Ca bioaccessibility and polyphenol content by as much as 19 and 22%, respectively (Krishnan et al., 2012). Microwave cooking by boiling also does not greatly improve the percentage of soluble or bioavailable Ca (Amalraj and Pius, 2015). This implies that HTST-based processing and microwave cooking methods do not favor improved bioaccessible Ca from finger millet. On the other hand, sprouting of finger millet improves the extractability of Ca and lowers antinutrients like phytate and tannins to an undetectable level after 4 days (Mbithi-Mwikya et al., 2000). Flour made from whole grain finger millet was found to have higher Ca content (325 mg/100 g) than those made from decorticated (222 mg/100 g) one (Hemanalini et al., 1980). Decortication is a process of removal of the seed coat matter which is responsible for lowering Ca bioaccessibility. It is interesting to note that processing by decortication significantly improved Ca bioavailability in rats and this was attributed to its low fiber and phytic acid content (Hemanalini et al., 1980). In an evaluation of various processing methods of finger millet on Ca bioaccessibility, seed decortication and malting were found to be the most efficient techniques (Krishnan et al., 2012). Decortication improves Ca bioaccessibility by 37.5%, in spite of lowering the total Ca content by 40%. This increase was attributed to a direct decrease in inhibitory contents present in the seed coat like phytic acid (31% reduction) and polyphenols (70% reduction). Malting, which involves germination and thermal treatment, also influences the bioaccessible Ca content of finger millet in a positive way (Platel et al., 2010; Krishnan et al., 2012). Again, this is because germination process greatly reduces the concentration of phytic acid and polyphenols. Fermented flour or sprouting followed by fermentation also showed marked enhancement in Ca availability (20%) with a concomitant decline in phytates, phenols, tannins, and trypsin inhibitor activity (Sripriya et al., 1997; Antony and Chandra, 1998; Makokha et al., 2002). The above examples establish beyond doubt that bioaccessibility of Ca from finger millet can be even further improved by simple processing methods which can be scaled up to industrial levels. These methods have potential to add values to both traditional as well as to contemporary value-added food products improving their edible and sensory properties (Hotz and Gibson, 2007; Shobana et al., 2013; Verma and Patel, 2013). ### Host (Extrinsic) Factors That Influence Ca Bioavailability Apart from the Ca bioavailability parameters, the capability to determine the effect on Ca status on target populations is another specific challenge. Host factors, such as age, gender, dietary patterns may show differential effects of finger millet-based diets on the net Ca contribution. These factors must be considered in controlled feeding community-based studies to determining the biological impact of biofortified crops. In the past, various attempts have been put together to assess the Ca bioavailability in vivo. Early nutrition studies have shown that rats fed with a diet composed of 70% finger millet retain 68% Ca (Giri, 1940). A further reduction of the finger millet content to 20–40% in diets contributed to increased Ca retention to 84–88% levels (Giri, 1940). This shows that even a low dietary component of finger millet is sufficient to maintain Ca availability because of its high Ca content. In fact, in a more recent study, Ca from finger millet had shown to have a better uptake as compared to commercial Ca supplementation tablet (Bhide et al., 2013). In this in vivo study, the serum Ca level of rats fed with finger millet extract and a finger millet based ready-to-drink formulation was >35% higher as compared to conventional Ca tablet supplemented group. However, for human metabolism studies, host factors like age and gender are important parameters to estimate daily requirement, intake and retention of dietary Ca. Many nutrition reports have estimated the contribution of finger millet for Ca homeostasis in humans. A study by Subrahmanyan et al. (1955) had found that a finger millet variety, H22, with Ca content 440 mg/100 g can on an average provide 3.4 g Ca/ day to healthy adult males aged 22–32 years. This amounted to Ca retention of 98 mg (approximately 3%) from a total daily intake of 3.4 g/day. This was higher than a brown bread or Ca carbonate fortified brown bread diet providing only 0.5–1.2 g Ca/day, respectively (McCance and Widdowson, 1942). It is recommended that diets should provide at least 200 mg/100 g of Ca to counteract the anticalcifying effect of phytic acid (McCance and Widdowson, 1942). Interestingly, 86% of phytate ingested from the fingermillet-based diet was hydrolysed during digestion and absorption process (Subrahmanyan et al., 1955). As most of the phytate is broken down during digestion, therefore, regular inclusion of finger millet in diet can efficiently maintain a positive Ca balance. In another study, young girls (aged 9–10) were fed on four different diets containing only rice, 75% rice + 25% finger millet, 50% rice + 50% finger millet and only finger millet as the primary cereal (Joseph et al., 1959). About 19% Ca was retained from just rice-based diets which increased to 22.5– 25.3% when a portion of rice was replaced with 25–50% finger millet. Therefore, finger millet can naturally contribute to boost the Ca status across ages evidently surpassing the other cereals. One serving of finger millet-based Ca-rich products, which were processed to increase Ca bioavailability, was shown to provide >0.2 g Ca contributing to 25% of Indian RDA of Ca for children and adolescents (Sanwalka et al., 2011). It is interesting to note that the Ca retention capacity of children seems to be much higher than adult subjects. This may be because growing age lowers digestibility and retention capacity of Ca and hydrolysis of antinutrients like phytate (Yoshida et al., 1983). It needs to be stressed here that most of these studies have been conducted more than 50 years ago. However, lifestyle and dietary patterns have drastically changed in recent years. Therefore, evaluating positive Ca impacts of finger millet diet across various groups needs to be measured keeping in view the current scenarios as well. In a case study in rural India, the dietary patterns of women self-help groups was assessed for their nutrient adequacy (Vijayalakshmi et al., 2010). The families usually consumed one portion of finger millet preparation two times a day along with one portion of pulses and vegetables. It was found that irrespective of the socio-demographic profile of the subjects (like age, education, family income, family size), Ca levels met the recommended RDA and their adequacy was attributed to regular finger millet inclusive diet. As no major replacement of diet is necessary, finger millet and its derived food products have the advantage to be more acceptable to the people. This makes it a more viable option to be effective providing adequate Ca intakes and prevent Ca deficiency. These reports provide an idea that designing food products from biofortified varieties of finger millet can easily supplement and add-on to the daily Ca intake across ages and genders with various dietary practices. Such information can allow the acceleration of finger millet biofortification programs. ### MAJOR CHALLENGES TO DEVELOP FINGER MILLET AS A MODEL FOR CALCIUM BIOFORTIFICATION In order to develop Ca biofortified finger millet, nutritionists must have available resources (superior Ca-rich varieties), a welldeveloped methodology to evaluate the bioaccessibility (in vitro and in vivo), awareness of the limiting factors (enhancers and inhibitors), and prior assessment of efficacy and effectiveness of Ca biofortified food. However, currently the development in this area is extremely limited and developing more nutritious varieties is challenging. Given that millet biofortification has recently been strategized, development of research programs for Ca-biofortified finger millet will have to address some arguments as discussed below. ### Challenges to Efficiently Utilize the Available Germplasm Resources and Genetic Diversity In recent years, various efforts have been made by geneticists and breeders to identify naturally occurring genetic diversity in finger millet. However, the major challenge at present is how these resources could be exploited to develop Ca-biofortified finger millet. Currently, finger millet genebanks across the globe conserve more than 37,000 accessions with India, Kenya, Ethiopia, Uganda, and Zambia housing the major collections (Vetriventhan et al., 2015). As of now, the entire genetic diversity present among the finger millet germplasm is available as small sets (core) and sub-sets (mini core) collections (Vetriventhan et al., 2015). Using these collections, 15 accessions were identified as most promising (3.86–4.89 g/kg) for further improving grain Ca content in cultivated finger millet (Upadhyaya et al., 2011). Recently, another core set of 77 germplasm of Indian and African origin has been formed using the base germplasm of finger millet 1,000 accessions (Chandrashekhar et al., 2012). In addition, finger millet composite collections (1,000 accessions) and a derived reference set (300 accessions) representing regionand race-based available diversity of the entire collection, is also available (Upadhyaya et al., 2005; Upadhyaya, 2008). Although, these large collections of finger millet germplasm serve as an ideal resource to be utilized in improving its Ca concentration, a majority of it remains largely underutilized for breeding high Ca finger millet varieties. Some of the main reasons for this lag are due to factors, such as weak and insufficient strategies for harnessing the useful genetic diversity available in these collections, barriers related to introduction and crossing of exotic germplasm, few pre-breeding programs to facilitate introgression of desirable nutrition quality into breeding lines and recirculation of same working collections by breeders (Dwivedi et al., 2009; Upadhyaya et al., 2014). Although such precious germplasm collections exist, sometimes there are also practical barriers associated with their availability for use. For example, restricted global exchange of accessions due to legal aspects of seed transfer agreements poses a limitation for verification of adaptability to multi-location or multi-environment trials (Nass et al., 2012). Even a dearth of trained finger millet breeders to meet the demand can be an issue for making use of this treasure. In addition, a huge range of diversity for grain Ca also exists within the gene pool of finger millet which remains to be exploited in targeted breeding. Two main gene pools exist for this species, namely Eleusine coracana sub-species africana (wild progenitor) and E. coracana sub-species coracana (domesticated cultivars/varieties and landraces (Agarwal and Maheshwari, 2016). Considerable variation in grain Ca contents has been observed in both the genepools. A detailed systematic analysis of grain Ca content in E. coracana sub species africana, from Ethiopia, revealed significantly higher Ca (515 mg/100 g) than the domesticated E. coracana sub species coracana from Kenya (401 mg/100 g) and India (375 mg/100 g) (Barbeau and Hilu, 1993). Many pieces of evidence for genotype effect in finger millet Ca content also exist. Vadivoo et al. (1998) found a large heritable genetic variation in relation to Ca content in 36 genotypes of finger millet, with white seeded varieties containing moderate levels of Ca. Based on their results, Malawi 1915 (486.7 mg/100 g Ca) and CO 11 (487 mg/100 g Ca) genotypes were proposed to be employed as crossing parents in breeding for the improvement of Ca content. Similarly, the dark red to very dusty red colored finger millet genotypes, CO 10, KM 1 and MI 302 sourced from the Dry zone Agricultural Research Station, Sri Lanka were shown to contain 240–250 mg% Ca (Ravindran, 1991). In another report, the white seeded finger millet varieties showed higher average Ca (329 mg%) than the brown seeded ones (296 mg%) (Seetharam, 2001). In spite of the extensive screening of finger millet germplasm for grain Ca content, the identified potential candidate genotypes have remained unused for developing higher Ca containing varieties breeding. At the same time, just selecting suitable donor lines for selective breeding based on variation in grain Ca content is not sufficient and may not even be successful as such variations may often be regulated at various other levels. Therefore, determination of genetic stability and adaptability of this trait across multiple environments is one of the prerequisite to develop effective strategies for breeding elite lines. However, a severe gap exists in our knowledge about accuracy by which genetic variation for Ca content can be reproduced by finger millet genotypes grown across various agro-ecological conditions. ### Challenges Associated with Breeding-Based Genetic Improvement of Finger Millet As finger millet is a naturally self-pollinating crop, artificial hybridisation by crossing of suitable parental lines is often a difficult task. Mass and pure-line selection practices have come in handy for inter-varietal improvement for grain yield, early maturity and disease resistance (Harinarayana, 1986; Table 2). For example, using pure line selection from the germplasm accession, finger millet culture WWN-25 has been released as a high yielding variety, GNN-7, for cultivation in Gujrat state of India (Patil et al., 2016). This is a promising development as this variety contains higher Ca (468.0 mg/100 g) than the national check variety VR-708 (398.0 mg/100 g) without compromising on the yield. However, optimum deployment of other breeding methods, such as recombination breeding, for generating stable hybrids, breeding progeny and inbred lines has been delayed due to challenging biparental cross, difficult emasculation and artificial hybridization in finger millet. To overcome these challenges, induced mutations, such as genetic male sterile systems (viz., INFM 95001 reported by ICRISAT; http://oar.icrisat.org/618/1/PMD\_71.pdf) have proved to be another efficient breeding tool for yield and disease resistance in finger millet. These systems and their subsequent breeding can be used effectively to increase the genetic variance by creating new recombinants and segregating populations by exploiting the genetic background. Therefore, developing genetic resources for finger millet, such as mapping populations, breeding lines and male-sterile mutant lines (Gupta et al., 1997; Krishnappa et al., 2009; Parashuram et al., 2011), deserves attention. Such material will be immensely valuable for tagging nutritional quality traits, especially grain Ca content, and thus facilitate genetic biofortification of finger millet. ### Underutilization of Available Genetic and Genomic Resources for Molecular Breeding Applications Efforts have been made to generate molecular markers for characterizing important traits like grain Ca and protein content and resistance to blast infection in finger millet. Genomic tools like SSR markers have helped to assess the range of genetic diversity for grain Ca content in various finger millet genotypes (Panwar et al., 2010; Nirgude et al., 2014; Yadav et al., 2014; Kumar et al., 2015a). Nine SSR markers derived from candidate Ca transporter and sensor genes have been found to be significantly associated with the Ca trait. They could serve as an important functional resource for the genetic improvement of finger millet's nutritional value through markerassisted breeding (MAB; Kumar et al., 2015a; Sharma et al., 2017). However, the inbreeding nature, limited recombination rates and a historical genetic bottle-neck during isolated domestication of this crop significantly impacts the extent of available genetic diversity in finger millet. Such loss of genetic diversity is a challenge for geneticists and breeders working with a limited number of finger millet accessions. Further, until recently, there has been no progress in application of the finger millet genetic map in trait mapping despite the assembly of the only molecular marker-based linkage map a decade ago (Dida et al., 2007; Srinivasachary et al., 2007). It still remains under-utilized for tagging and identification of genes/quantitative trait locus (QTL) governing grain Ca content probably due to an insufficient number of informative markers. The unavailability of sufficient markers and genome sequence information in finger millet has resulted in limited breeding efforts for nutritional improvement. Nonetheless, advances #### TABLE 2 \| Modern finger millet varieties released in the last decade. #### TABLE 2 \| Continued (Continued) ### TABLE 2 \| Continued in large-scale genomics technology have now streamlined production of genome-wide markers which can be used for large-scale identification of candidate genomic loci. This advancement has also been capitalized to generate single nucleotide polymorphism (SNP) markers in finger millet using genotyping-by-sequencing (GBS; Kumar et al., 2016b) and Roche 454 and Illumina sequencing (Gimode et al., 2016). Inspite of the low level of polymorphisms in cultivated finger millet genotypes (Salimath et al., 1995), these SNPs may provide some explanation for variation in grain Ca content among finger millet genotypes. However, before their utilization, it is crucial to differentiate true SNPs among different genotypes from the homeologous SNPs within an individual genotype due to allotetraploidy (AA and BB sub-genomes) of finger millet. Advances in cereal genomics have elaborated that genomic level similarities are conserved in the relative physical positions across species both on a fine scale (co-linearity) as well as on a chromosomal scale (synteny). For example, comparative mapping of finger millet to rice has shown a fairly high level of collinearity among these crops (Srinivasachary et al., 2007). The availability sequence information for several members of the grass family is now assisting in the development of inter-species molecular markers and gene discovery in unexplored crop genomes, such as finger millet (Wang et al., 2005; Kalyana Babu et al., 2014a,b). Until finger millet whole genome sequence becomes available, such comparative genomics approaches can benefit finger millet improvement programs. However, identification of common quantitative trait loci (QTLs) that control grain Ca content among cereals remains to be explored. This will further facilitate identification of orthologous regions and transfer of genetic information across species for improving Ca content of other millets and nonmillets. ### Inadequate Understanding of Ca Homeostasis Mechanisms in Finger Millet Emphasizing on the molecular status of Ca accumulation in finger millet grains, various genes have recently been identified (Sood et al., 2016). More recently, a molecular model for Ca transport from soil to seed has been proposed (Sharma et al., 2017). This hypothetical model is based upon genes that are differentially expressed in contrasting finger millet cultivars (Mirza et al., 2014) or grain filling and developing spike transcriptome studies in this crop (Singh et al., 2014, 2015; Kumar et al., 2015b). These genes correspond to Ca sensing and binding, Ca transport and seed storage, such as, type IIB ATPase, Ca2+/H<sup>+</sup> antiporter (CAX1), two pore channel (TPC1), calmodulin (CaM), CaM-dependent protein kinases (CaMK1 and CaMK2) and 14-3-3 (Table 3). High Ca accumulation in finger millet has been mainly attributed to the Ca sensor genes which have been proposed as candidates for targeted Ca enhancement in finger millet varieties (Singha et al., 2016). Alteration in the genes expression levels only indicates a pattern among the contrasting genotypes which does not always validate the translated protein products involved in Ca accumulation. A few recent studies have characterized accumulation of Cabinding protein (calreticulin) and CaM protein during grain filling stages of finger millet (Kumar et al., 2014; Singh et al., 2016). Unfortunately, large-scale protein profiling to identify the complete set of proteins involved in finger millet Ca homeostasis is still unavailable. Even though the past and current developments have generated a huge wealth of transcriptomic datasets linked with the Ca uptake, translocation and accumulation in finger millet, without functional characterisation, these candidate genes are merely speculation. With a lack of evidence for efforts being made to functionally characterize these genes/proteins, it is possible that these potential candidates may not have expected TABLE 3 \| List of important genes identified for calcium uptake, transport and storage in finger millet. impact on enhancing grain Ca content. While, it is practically impossible to functionally characterize all genes linked with Ca acquisition, sensing and accumulation, transgenic technology along with progress in gene editing (such as CRISPR-Cas and TALEN) can aid in a better functional understanding of many candidate genes. Such advancements in gene editing technology along with well-established genetic transformation protocols for generation of transgenic finger millet (Kumar et al., 2016a) can significantly improve our understanding of this process. Besides this, the role of other factors (for example, hormones, root morphological traits, endophytes, soil fertility, evapotranspiration pull, translocation distance of Ca) on Ca homeostasis in finger millet remains unknown. This is a crucial question as the soil-plant interactions substantially affect the available proportion of micronutrient to the roots. In this context, soil medium supplemented with growth promoting rhizobacterium (Azotobacter, Azospirillium, phosphorus solubilizing bacteria) and vesicular-arbuscular mycorrhizal (VAM) fungi are generally practiced to enhance the grain yield and growth in finger millet (Ramakrishnan and Bhuvaneswari, 2014; Thilakarathna and Raizada, 2015). VAM are also known to increase the content and uptake of minerals, such as phosphate, nitrogen, zinc and copper (Tewari et al., 1993). The nutrient supply to the plant can be augmented either by efficiently mobilizing nutrient forms available in the soil or by extending the nutrient absorption surface by designing better roots system. However, how these associations impact Ca absorption and uptake in finger millet has received little experimental consideration. Further, the relationship between growth environments and climates may also alter xylem water flow, thus indirectly determining Ca distribution. So far, the established mechanisms of Ca accumulation in finger millet have been developed on the basis of controlled growth conditions. However, under field conditions, the unique soilroot interaction can influence the Ca-sensing and transport differently. In addition, any confounding effects of agronomic traits, such as vegetative growth, yield, stress resilience and disease resistance on grain Ca accumulation are not clearly established. Therefore, to develop finger millet as a model for Ca biofortification, we need a comprehensive understanding of the mechanisms and other factors that may influence this trait. ### Potential of Next Generation Sequencing (NGS) For Improved Finger Millet Varieties Enormous progress has been made in the genomics technology through application of high-throughput, economical and quicker next generation sequencing (NGS) platforms. Extending the benefits of NGS to finger millet, a recent effort of de novo sequencing has allowed whole genome sequence assembly covering approximately 82% of total estimated genome size (Hittalmani et al., 2017). Evidence of higher collinearity with foxtail millet and rice as compared to other Poaceae species, and the available genome sequencing information may help allele discovery and candidate gene identification for agronomically important traits (Hittalmani et al., 2017) leading to faster development of improved varieties. In addition, GBS, which is a NGS platform-based highly multiplexed genotyping system, has also been applied for SNP generation (Kumar et al., 2016b). Thus, now it is feasible to generate a higher density of markers by genotyping core collections of finger millet thereby increasing the level of genetic diversity explored. This is crucial for a predominantly self-fertilized crop like finger millet because it is expected to have low recombination rates and high linkage disequilibrium (LD) which would otherwise narrow the genetic diversity. Thus, the more genetically diverse populations in finger millet core collections, together with the huge amount of relevant marker information generated through NGS platforms can directly contribute to improved mapping resolution of traits, such as Ca content through genome-wide association studies (GWAS). By statistically reconnecting variation in grain Ca content back to its underlying genetic polymorphism, it is possible to identify functional common variants in LD and genomic regions where major-effect genes and QTLs (that serve as the targets of marker-assisted selection) are located. The GWAS studies can confirm previously identified genes involved in Ca homeostasis mechanisms as well as spot putative novel candidates. However, the efficiency of GWAS depends upon accurate grain Ca content phenotyping data over multilocation/multi-year trials. An extension of MAS, genomic selection (GS) is an upcoming methodology in the area of genomics-assisted breeding (Meuwissen et al., 2001). In this approach, genomewide marker genotype data along with available phenotypic data for a tested (reference/training) population are used to predict the performance of an untested (breeding) population based on genomics estimated breeding values (GEBV). Thus, instead of identifying few large-effect loci associated with Ca content, the GEBV model can more accurately predict the expected phenotype of a broader breeding population. This significantly reduces the time and costs associated with phenotyping a trait like grain Ca content. Finger millet enjoys the availability of germplasm resources, such as the core collections, which can be utilized as test populations to build genomic prediction models. As GS eliminates the need for previous identification of major QTLs and their use in selection, it can substantially speed up the genetic gain in this "orphan" crop. Another advantage is that if GEBVs are efficiently evaluated, finger millet breeders can make appropriate selection choices even earlier in the program, thereby significantly saving time on the generation cycle (Heffner et al., 2010). However, the applicability of GS in finger millet and selection of superior genotypes are dependent upon precise measurement and heritability of Ca content, sufficient marker density, the extent of LD decay, effective design of training population and its genetic relationship with the breeding population (Varshney et al., 2014). Nevertheless, NGS can contribute in exploring the depth and breadth of genetic diversity across germplasm sets bringing forward a huge wealth of genetic information. This will eventually lead to a new horizon for finger millet Ca biofortification. ### FUTURE PROSPECTS AND CONCLUSIONS By virtue of its health benefitting properties and environmental sustainability, a traditional but less popular crop like finger millet offers excellent opportunities for biofortification breeding. A foremost priority from geneticists and breeders viewpoint is capturing and utilizing genetic diversity for Ca content in the elite finger millet gene pools (for example, by bringing new sources of variation through rare and unique alleles). For trapping such useful variations, advances in the next generation sequencing technology must be utilized in generating sufficient number of markers for characterizing marker-trait associations and genomics-assisted breeding. With the implementation of such high-throughput approaches, it will be much easier to investigate the genetic architecture of this trait through comparative mapping in other millets and non-millet species. Mining of markers tightly linked to other traits governing grain Ca content and discovery of underlying genes can be an alternate strategy to develop high Ca finger millet varieties through traditional or modern breeding approaches and transformation-based methods. For example, rather than just arbitrarily increasing grain Ca content, future direction should target improvement in efficiency to mobilize, acquire, transport and store Ca in more bioavailable forms in the edible portions. From the health perspective, at this point, we almost completely lack the understanding of interplay among grain Ca, other micronutrients and antinutrients metabolism in human body. Any potential risks of reducing the antinutrient content in the grains should be evaluated with a view of their longterm effects on human health. Therefore, the focus should be on demonstrating finger millet's bio-efficacy in order to monitor any potential negative trade-offs and unintended effects. In addition, experimental confirmation of in vivo Ca bioavailability needs to be cautiously attempted through designing pilot feeding studies for vulnerable groups (like children, nursing or post-menopausal women). Although the biological effects of improved finger millet may be relatively modest, it will have potential benefits ### REFERENCES in improving healthcare cost savings by reducing the risk of osteoporotic fractures and DALYs lost. While agronomic factors can influence acceptability of the improved finger millet varieties by farmers, other parameters are important from the view of consumer acceptance. The extent of Ca recommended dietary allowance proportion as well as the sensory satisfaction supported by finger millet is essential in terms of developing food products keeping in line with recent lifestyle changes. Even after successful Ca biofortification of finger millet, its introduction and success as a functional food still entails knowledge of adequate food processing strategies (to minimize the nutrient loss) and study of consumer preferences. Further, communication support and the creation of market demand for its value-added products will be necessary. Therefore, a multi-disciplinary research approach, incorporating nutrition, health, agriculture, along with policy and market research, is needed to ensure the impact of high Cabiofortified finger millet. Overall, it is worthwhile to conclude that finger millet biofortification will improve the quality of life for both the rural subsistence farming families as well as the consumers. ### AUTHOR CONTRIBUTIONS SP and RY conceptualized the manuscript. SP wrote the manuscript. JK, PS, HO, and RY assisted and edited the manuscript. SP, JK, PS, and RS contributed in critically revising the draft and updating the manuscript for publication. ### ACKNOWLEDGMENTS The authors wish to express their thanks to IBERS, UK for the support in writing this manuscript. IBERS receives strategic funding from BBSRC. SP acknowledges Marie Skłodowska-Curie Individual Fellowship from Horizon 2020 of European Commission (Project 657331; CaMILLET). We would also like to acknowledge the editor and reviewers whose constructive comments and suggestions helped to improve the manuscript. on Biofortification: Getting Nutritious Foods to People. March 31–April 2, 2014 (Kigali). of bioactive compounds from fruits and vegetables: a review. Compr. Rev. Food Sci. Food Saf. 13, 155–171. doi: 10.1111/1541-4337.12049 in differential accumulation of calcium in developing grains. Appl. Biochem. Biotechnol. 172, 2955. doi: 10.1007/s12010-013-0714-0 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Puranik, Kam, Sahu, Yadav, Srivastava, Ojulong and Yadav. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. # Genome-wide Identification, Characterization, and Expression Analysis of PHT1 Phosphate Transporters in Wheat Wan Teng<sup>1</sup>† , Yan-Yan Zhao<sup>1</sup>† , Xue-Qiang Zhao<sup>1</sup> , Xue He<sup>1</sup> , Wen-Ying Ma<sup>1</sup> , Yan Deng<sup>2</sup> , Xin-Ping Chen<sup>3</sup> and Yi-Ping Tong<sup>1</sup> \* <sup>1</sup> The State Key Laboratory for Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, <sup>2</sup> Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, China, <sup>3</sup> Research Center of Resource, Environment and Food Security, China Agricultural University, Beijing, China #### Edited by: Raul Antonio Sperotto, Centro Universitário UNIVATES, Brazil ### Reviewed by: Stefano Cesco, Free University of Bozen-Bolzano, Italy Hatem Rouached, Institut National de la Recherche Agronomique (INRA), France Soren K. Rasmussen, University of Copenhagen, Denmark \Correspondence: Yi-Ping Tong [email protected] †These authors have contributed equally to this work. #### Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 11 February 2017 Accepted: 27 March 2017 Published: 11 April 2017 #### Citation: Teng W, Zhao Y-Y, Zhao X-Q, He X, Ma W-Y, Deng Y, Chen X-P and Tong Y-P (2017) Genome-wide Identification, Characterization, and Expression Analysis of PHT1 Phosphate Transporters in Wheat. Front. Plant Sci. 8:543. doi: 10.3389/fpls.2017.00543 The PHT1 family of phosphate (Pi) transporters mediates phosphorus (P) uptake and re-mobilization in plants. A genome-wide sequence analysis of PHT1 genes in wheat (Triticum aestivum) was conducted, and their expression locations and responses to P availability were further investigated. We cloned 21 TaPHT1 genes from the homologous alleles at TaPHT1.1 to 1.10 through screening a BAC library and amplifying genomic sequences. The TaPHT1 transporters were clustered into five branches in the phylogenetic tree of PHT1 proteins, and the TaPHT1 genes from a given branch shared high similarities in sequences, expression locations, and responses to P availability. The seven tested PHT1 genes all showed Pi-transport activity in yeast (Saccharomyces cerevisiae) cells grown under both low Pi and high Pi conditions. The expression of TaPHT1.1/1.9, 1.2, and 1.10 were root specific. The expression of these TaPHT1 genes at flowering positively correlated with P uptake after stem elongation across three P application rates and two wheat varieties in a field experiment. Therefore, modification of PHT1 expression may improve P use efficiency in a broad regime of P availability. Keywords: wheat (Triticum aestivum), PHT1 genes, genome-wide analysis, phosphate transporter, phosphorus uptake, phosphate-starvation response ## INTRODUCTION Phosphorus (P) is one of the essential macronutrients for plant growth and development, and it takes part in cellular macromolecules, energy transfer reactions, and cellular metabolism. Efficient acquisition of phosphate (Pi) from soil combined with efficiency translocation of Pi within plants is essential for plants to maintain adequate levels of cellular Pi necessary for normal function (Raghothama and Karthikeyan, 2005). Although total P in soils is abundant, the soluble phosphate (Pi) is often low (Bieleski, 1973; Rausch and Bucher, 2002), and therefore plants often encounter a scarcity of Pi in soils of both agricultural and natural systems (Raghothama, 1999, 2000). As there is a large difference between Pi levels in plant cells (mM) and soil solution (µM), plants need to acquire Pi against a steep concentration gradient across the plasma membrane (Smith et al., 2003; Raghothama and Karthikeyan, 2005). The transmembrane transport of Pi from soils into plant cells requires a high-affinity, energy-driven transport mechanism (Smith et al., 2003). The PHT1 family of plant Pi transporters is assumed to play the predominant roles in this transmembrane transport process. These proteins are characterized by 12 membranespanning domains which are similar to PHO84, a high-affinity Pi transporter from yeast (Saccharomyces cerevisiae) (Muchhal et al., 1996; Rausch and Bucher, 2002). There are four PHOSPHATE TRANSPORTER (PHT) families in plants: PHT1, PHT2, PHT3, and PHT4 which are located on plasma membrane, plastid inner membrane, mitochondrial inner membrane, and Golgi-compartment, respectively (Lopez-Arredondo et al., 2014). Under the stress of P-starvation, the expression of PHT1 genes are strongly induced to increase the ability of the roots in acquiring P from soils and remobilize P within plants (Smith et al., 2003; Raghothama and Karthikeyan, 2005). A large number of PHT1 transporters have been identified in many plant species and show differences in expression locations and affinities for Pi (Nussaume et al., 2011). Nine PHT1 genes in Arabidopsis (Arabidopsis thaliana) have been identified. AtPHT1.1 and AtPHT1.4 are highly expressed at the root–soil interface, including the epidermis, root hair cells, and the root cap under low P conditions (Mudge et al., 2002), and they are the major genes responsible for Pi acquisition by roots in both high and low P supplies (Misson et al., 2004; Shin et al., 2004; Catarecha et al., 2007). AtPHT1.8 and AtPHT1.9 are likely to act sequentially in the interior of the plant during the root-to-shoot translocation of Pi and are involved in rootto-shoot translocation of Pi (Lapis-Gaza et al., 2014). There are 13 PHT1 members in the rice (Oryza sativa) genome, and some of them have been functionally characterized, including OsPHT1.1 (Sun et al., 2012), OsPHT1.2 and OsPHT1.6 (Ai et al., 2009), OsPHT1.4 (Ye et al., 2015), and OsPHT1.8 (Jia et al., 2011; Li et al., 2015). For example, OsPHT1.6 is expressed in both epidermal and cortical cells of the younger primary and lateral roots and encodes as a high-affinity transporter with a broad role in Pi uptake and translocation throughout the plant, whereas OsPHT1.2 is localized exclusively in the stele of primary and lateral roots and functions as a low-affinity transporter responsible for Pi translocation (Ai et al., 2009). Barley (Hordeum vulgare) is a close relative to wheat (Triticum aestivum). To date, 11 PHT1 genes have been reported in barley. The HvPHT1.1 and HvPHT1.2 promoters drive the expression of β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes in epidermal and cortex cells as well as vascular tissues of roots (Schunmann et al., 2004). When expressed in Xenopus laevis oocytes, HvPHT1.1 is confirmed to be a high-affinity transporter with a very low K<sup>m</sup> value (1.9 µM) for Pi transport (Preuss et al., 2011). The expression locations and K<sup>m</sup> value for Pi transport indicate the possible role of HvPHT1.1 in P uptake. HvPHT1.6 is expressed in both roots and shoots (Huang et al., 2008). Also, it is highly expressed in old leaves compared to young leaves, especially in the leaf phloem tissue (Rae et al., 2003). HvPHT1.6 shows the linear transport activity for Pi-stimulated inward current over a concentration range of 5 to 30 mM in Xenopus laevis oocytes (Preuss et al., 2010). These results suggest that HvPHT1.6 function as a low-affinity Pi transporter responsible for Pi remobilization in the whole plant. Huang et al. (2011) investigated the expression of PHT1 genes and its relationship with P acquisition efficiency and P utilization efficiency (the amount of biomass produced per unit of acquired P) in four barley genotypes. They did not find a clear pattern in the expression of the four HvPHT1.1 paralogs (HvPHT1.1, 1.2, 1.9, 1.10) among the four barley genotypes, but observed that the expression of HvPHT1.3 and 1.6 positively correlated with P utilization efficiency. HvPHT1.8 and HvPHT1.11 (known as HvPT11) have been demonstrated to be specifically activated by arbuscular mycorrhizal (AM) fungi (Glassop et al., 2005; Sisaphaithong et al., 2012), indicating their possible roles in the mycorrhizal pathway of Pi uptake. Wheat is one of the most important crops. However, limited attempts have been made to dissect the role of Pi transporters in wheat (Secco et al., 2017). Davies et al. (2002) isolated the first full-length sequence of a wheat PHT1 gene (TaPHT1.10-U, formerly known TaPT2) and partial clones of several other putative PHT1 genes. TaPHT1.10-U was induced by P-deficiency in roots, and had higher transcript abundance in P-efficient wheat varieties than in inefficient ones (Davies et al., 2002). In yeast, TaPHT1.10-U can complement highaffinity phosphate transporter gene PHO84 function (Zeng et al., 2002) and shows an apparent mean K<sup>m</sup> of 23.6 µM Pi (Guo et al., 2014). Overexpression of TaPHT1.10-U increases plant dry weight and Pi acquisition, whereas knock-down of TaPHT1.10-U has the opposite effect (Guo et al., 2014). These results suggest that TaPHT1.10-U functions as a high-affinity Pi transporter and mediates Pi uptake. A recent study observed that TaPHT1.12-7A (former name TaPHT1.4) was root-specific and P-deficiency inducible. Yeast complement analysis showed that TaPHT1.12-7A encodes a high-affinity Pi transporter with an apparent K<sup>m</sup> of 35.3 µM. Overexpressing TaPHT1.12-7A significantly improves growth traits and accumulates more Pi than the wild-type plant and those with downregulated TaPHT1.12-7A expression (Liu et al., 2013). A recent study also revealed the relationships between PHT1 expression and P use efficiency in wheat (Aziz et al., 2014). The highly P-efficient wheat cultivar Chinese 80-55 has a higher Pi acquisition in the presence of Pi and accumulates higher Pi concentrations in all organs upon Pi withdrawal compared with the less-efficient cultivar Machete. These differences correlate with differential organspecific expression of Pi transporters TaPHT1.10-4A (reported as TaPHT1.2, GenBank: AY293828), TaPHT1.6-5A (reported as TaPHT1.5, GenBank: AF110180) and TaPHT1.4-5B (reported as TaPHT1.8, GenBank: AK333026) (Aziz et al., 2014). Shukla et al. (2016) found that aleurone accumulates more Pi with higher expression of TaPHT1 genes compared to endosperm. TaPHT1.8- 6A (known as TRIae; Pht1; myc, Glassop et al., 2005, GenBank: AJ830009), TaPHT1.11-4A (known as TRIae; Pht1; 12, GenBank: AB753271), TaPHT1.11-4B (known as TRIae; Pht1;11, GenBank: AB753270), and TaPHT1.11-4D (known as TRIae; Pht1; 10, GenBank: AB753269) have been found to be induced by AM fungi (Glassop et al., 2005; Sisaphaithong et al., 2012). Although expression of some PHT1 genes has displayed correlation with P use-related traits in wheat and its close relative barley under controlled conditions, an on-farm field-scale investigation is required to explore the PHT1 genes contributing to P uptake and utilization, as the response of PHT1 genes to P supply level under controlled conditions greatly differed from that under field conditions. Our recent study showed that the expression of TaPHT1.1, 1.2, 1.9, and 1.10 in roots at the flowering stage under low P conditions was lower than that under high P conditions in a field experiment (Teng et al., 2013). The inhibition of these four wheat genes by P-deficiency could be, at least partially, explained by the upregulated AM colonization under P-deficiency, considering that AM colonization has been found to inhibit the response of HvPHT1.1 and HvPHT1.2 to P deficiency in barley (Glassop et al., 2005). In this study, we aimed to identify the sequences of PHT1 genes in the whole genome of wheat, and to analyze the correlation between the PHT1 expression and P uptake under field conditions. We isolated 21 full length sequences of PHT1 genes in wheat, and further analyzed their functions, expression location and response to P supply level. We observed that the expression of TaPHT1.1, 1.2, 1.9, and 1.10 in roots at the flowering stage contributed to P uptake of different wheat varieties under field conditions. ### MATERIALS AND METHODS ### Wheat Varieties The winter wheat (Triticum aestivum) variety Xiaoyan 54 was commercially released in 2000, and was used to isolate TaPHT1 sequences, and to analyze gene expression location and response to P availability. The winter wheat varieties Kenong 9204 (KN9204) and Shijiazhuang 8 (SJZ8) were commercially released in 2003, and were used in the field experiments to analyze the relationship between TaPHT1 expression and P uptake. ### Isolation of PHT1 Pi Transporters in Wheat To isolate PHT1 sequences from the wheat variety Xiaoyan 54, we performed BAC library screening and genomic sequence amplification by using the primers in Supplementary Table S1. After several rounds screening the BAC library of Xiaoyan 54 (Dong et al., 2010), we obtained 28 BAC clones which contained PHT1 genes. These BAC clones were sequenced commercially by using a Roche/454 GS-FLX Titanium System (Roche Diagnostics, Germany) at SinoGenoMax Co., Ltd. (Chinese National Human Genome Center, Beijing, China). The resultant sequences were examined for the promoter and protein-coding sequences of PHT1 genes, and consequently the primers were designed to isolate the coding regions of the PHT1 genes in these BAC clones. The PCR products amplified from BAC clones and genomic DNA were sub-cloned into a pMD18-T Vector (Takara Bio, Dalian, China), and then sequenced commercially at SinoGenoMax Co., Ltd. The putative cis-elements in the promoters were predicted by RSAT::Plants software<sup>1</sup> . We used the neighbor-joining method to generate a phylogenetic tree of PHT1 proteins from wheat, Triticum urartu, Aegilops tauschii, barley, maize (Zea mays), rice, and Arabidopsis, and the phylogenetic tree was drawn using MEGA 5.0 (Tamura et al., 2011). Sequence alignment was performed by DNAMAN6.0 (Lynnon BioSoft, San Ramon, CA, USA). #### <sup>1</sup>http://floresta.eead.csic.es/rsat/ ### Functional Complementation Assay of Pi Transporters in Yeast The yeast manipulations were performed as previously described (Ai et al., 2009). For the complementation assay, the coding sequences of the TaPHT1 genes were amplified by PCR and subcloned into the yeast expression vector p112A1NE to create TaPHT1-p112A1NE constructs. These constructs and the empty vector p112A1NE were transformed into the yeast Pi uptakedefective mutant MB192 (Bun-Ya et al., 1991). Because the PHT1 transporters are members of the H+/Pi symporter family, we firstly evaluated the optimal pH value for the growth of the transformed and control yeast strains. After measuring the optical density of the yeast cell lines at pH values ranging from 4 to 8 in yeast nitrogen base (YNB) liquid medium, we observed that the optimal pH value for most of the yeast mutant cells carrying TaPHT1s was 6, whereas the optimal pH value for the wild-type ranged from 4 to 6. Therefore, the pH value was set to 6 in the subsequent studies. To measure the kinetic growth profiles of the yeast strains, the yeast cells were grown in YNB liquid medium to the logarithmic phase (when the absorbance at 600 nm was 0.8), and were then harvested and washed in Pi-free YNB medium. Then, the yeast cells were grown at 30◦C for 24 h in the YNB liquid media containing 200 µM Pi (high Pi) and 20 µM Pi (low Pi). The absorbance at 600 nm (OD600) was recorded every 6 h. MB192 and p112A1NE were kindly provided by Prof. Shubin Sun from Nanjing Agricultural University, Nanjing, China. ### Plant Growth Conditions A hydroponic culture and three field experiments were conducted. The winter wheat variety Xiaoyan 54 was used in the hydroponic culture. The nutrient solution and growth conditions of the hydroponic culture were described by Wang et al. (2013). The seedlings, after 6 days of germination, were grown in nutrient solutions that contained 200 µM Pi (high P) or 5 µM Pi (low P). The plants were grown at 20◦C for 3 weeks, and the roots and shoots were collected separately for gene expression analysis. The field experiment in the experimental station of the Institute of Genetics and Developmental Biology in Beijing was carried out in the 2012–2013 growing season. The plant density and P fertilizer treatments was described by Wang et al. (2013). Briefly, the low P and high P treatments, i.e., 0.0 g m−<sup>2</sup> and 13.5 g m−<sup>2</sup> of P as calcium superphosphate, respectively, were applied before sowing. The seeds of Xiaoyan 54 were sown at the end of September in 2012. At the re-greening stage (March 18, 2013), the roots in 0–30 cm depth soil and shoots were collected separately. At the flowering stage (May 3, 2013), the stems, spikes, flag leaves, and aging leaves (top third leaf) were sampled. At the grain filling stage (14 days after flowering), the stems, grains, flag leaves, and aging leaves were collected. In each sampling time, 10 plants were randomly selected in each of the three replications. The plant samples were stored at −80◦C for gene expression analysis. Two field experiments at the Quzhou Experiment Station (36.5◦ N 115.0◦ E, 40 m above sea level) of the China Agricultural University have been described by Teng et al. (2013). These two experiments were conducted in the 2009–2010 growing season (referred as the 2010 field experiment) and the 2010–2011 growing season (referred as the 2011 field experiment). The data were collected from the winter wheat varieties KN9204 and SJZ8 at the P application rates 0, 100, and 400 kg ha−<sup>1</sup> of P as calcium superphosphate (referred as P0, P100, and P400, respectively). The data for P use-related traits and expression levels of TaPHT1 genes in KN9204 have been reported by Teng et al. (2013). The P application rates P0, P100, and P400 represented deficient, optimal, and excessive P supply, respectively (Teng et al., 2013). ### RNA Extraction and Quantitative Real-time PCR Total RNA extraction and real-time quantitative reverse transcription PCR (qRT-PCR) were performed according to the methods of Teng et al. (2013). The primer sequences are listed in Supplementary Table S2. The gene expression levels were normalized to the internal control of TaActin. ### Measurement of Total P Concentration in Plant Samples To determine plant total P, dried samples were milled and subsequently digested with concentrated H2SO<sup>4</sup> and H2O<sup>2</sup> using the molybdate-blue colorimetric method (Murphy and Riley, 1962). ### Statistical Analysis The SPSS statistical software (SAS Institute, Cary, NC, USA) was used to perform analysis of variance using one-way analysis of variance (ANOVA). Comparisons of means were performed using Duncan's multiple range analysis test and paired samples t-test (α = 0.05). ### RESULTS ### Sequence Analysis of PHT1 Transporters in Wheat We cloned 21 TaPHT1 genes from common wheat through screening a BAC library of Xiaoyan 54 and amplifying genomic sequences (Supplementary Table S3). None of these genes contained intron, 19 of them contained full length ORFs, and their deduced protein sequences varied from 521 to 539 amino acids (Supplementary Table S3). One nucleotide deletion occurred at 368 bp downstream of the start codon in TaPHT1.10- 4B and thus resulted in a frame shift mutation, and TaPHT1.9-4A had a premature stop codon mutation at 810 bp downstream of the start codon, but this premature stop mutation was not found in the Chinese spring. We mapped the cloned TaPHT1s on chromosomes by sequence analysis of BAC contigs and the reference sequence of Chinese spring<sup>2</sup> . The five clones, BAC48, BAC470, BAC674, BAC1217, and BAC1779, formed a BAC contig which contained TaPHT1.1-4B, 1.2-4B, 1.9-4B, and 1.10-4B (Supplementary Figure S1A). TaPHT1.9-4B and 1.2-4B matched with the sequences from 210,450 to 212,013 bp and from 299,422 to 300,999 bp in the scaffold TGACv1\_scaffold\_320302\_4BL, respectively (Supplementary Figure S1B), Therefore TaPHT1.1- 4B and 1.10-4B were assigned to chromosome 4B. Further sequence analysis showed that the 1011 bp fragment from 220136 to 221146 bp and the 1316 bp fragment from 258319 to 259634 bp of the scaffold TGACv1\_scaffold\_320302\_4BL matched with TaPHT1.10-4B and 1.1-4B, respectively, but both fragments had low sequence quality. The former fragment contained 585 unknown nucleotides, and the later fragment contained 1004 unknown nucleotides; this was possibly why these two fragments were not annotated. TaPHT1.10-4B also showed 99.7% of sequence identity with the sequence from 1 to 1465 bp in the scaffold TGACv1\_scaffold\_684896\_U (Overlapping gene TRIAE\_CS42\_U\_TGACv1\_684896\_AA2159320, Supplementary Table S3). TaPHT1.10-4B from Xiaoyan 54 seemed to be the allele of TRIAE\_CS42\_U\_TGACv1\_684896\_AA2159320 from Chinese spring, as both genes had the nucleotide deletion at 368 bp downstream of the start codon. Genome-wide analysis of the genome sequence in Triticum\_aestivum\_CS42\_TGAC\_v1 assembly for Chinese spring<sup>2</sup> totally identified 32 Gene IDs for TaPHT1 (Supplementary Table S3). The Gene ID TRIAE\_CS42\_4BL\_ TGACv1\_320302\_AA1034400 matched with TaPHT1.2-4B and TaPHT1.9-4B; and no Gene ID was found to match with TaPHT1.1-4B and TaPHT1.10-U cloned in the current study or TaPHT1.11-4B (former name TRIae; Pht1;11) cloned by Sisaphaithong et al. (2012). Therefore, we identified a total of 36 TaPHT1 genes (Supplementary Table S3). The 31 genes of TaPHT1.1-TaPHT1.11 were named according to their similarity with barley PHT1 transporters and chromosome location, and the remaining five genes were sequentially named TaPHT1.12, TaPHT1.13, and TaPHT1.14, together with the chromosome location (Supplementary Table S3). The TaPHT1 genes were unevenly distributed on the chromosomes, as there were 17 and 8 PHT1 genes on the chromosomes of homologous group 4 and 5, respectively (Supplementary Table S3). This uneven distribution was mainly due to the PHT1 clusters on the chromosomes of these homologous groups. For example, we found five TaPHT1 genes (TaPHT1.5-4B, TaPHT1.1-4B, 1.2-4B, 1.9-4B and 1.10-4B) within a 150-kb region on the long arm of chromosome 4B (Supplementary Figure S1). The scaffold TGACv1\_scaffold\_407907\_5BL on the long arm of chromosome 5B conferred TaPHT1.3-5B and TaPHT1.4-5B within an approximate 18-kb region (Supplementary Table S3). We also cloned the promoter sequences of 10 TaPHT1 genes, and all these promoters were found to contain several putative Pi-starvation response regulator PHR1 binding cis-element P1BS and WRKY transcription factor binding element W-Box (Supplementary Figure S2). We calculated the relatedness of TaPHT1s using the ClustalX 2.1 software, with the results suggesting that the protein sequence identities ranged from 46 to 99%. The highest identities were found between the protein sequences of TaPHT1.1/1.2/1.9/1.10, and for that of TaPHT1.3/1.4. There were more than 98% of protein sequence identities between each other of the <sup>2</sup>http://plants.ensembl.org/Triticum\_aestivum/Info/Index homologous alleles at a given TaPHT1 locus from genomes A, B, and D (e.g., TaPHT1.8-6A, -6B and -6D). A neighborjoining tree was constructed using a multiple sequence alignment according to TaPHT1 proteins and the PHT1 sequences from Triticum urartu, Aegilops tauschii, barley, maize, rice, and Arabidopsis (Supplementary Table S3). TaPHT1.10-4B was not included in the phylogenetic analysis, as it contained a frame shift mutation. The 35 TaPHT1s were clustered into five of the six branches (Figure 1). Branch I only contained PHT1s from Arabidopsis. TaPHT1.13-2A, TRIurPHT1.13, and OsPHT1.4/1.5 formed Branch II. The six TaPHT1.6/1.7 genes fell into Branch III which contained HvPHT1.6/1.7 and OsPHT1.6/1.7. The nine TaPHT1.3/1.4/1.5 genes belonged to Branch IV, and showed a close relationship with HvPHT1.3/1.4/1.5. The nine TaPHT1.1/1/2/1/9/1.10 genes belonged to Branch V, and they closely related to HvPHT1.1/1.2/1.9/1.10 and OsPHT1.1/1.2/1.3. The 10 TaPHT1.8/1.11/1.12/1.14 genes were grouped into Branch VI, which contained the AM fungiinducible PHT1s from cereals as well as AtPHT1.6/1.8/1.9 from Arabidopsis. ### Analysis of Pi Transport Activities of TaPHT1s in a Yeast Strain Defective in Pi Uptake We analyzed the Pi transport activities of TaPHT1.1-4D, 1.10-4A, 1.4-5D, 1.5-4A, 1.6-5D, 1.7-4D, and 1.8-6B genes using the yeast mutant MB192 strain (pho84 mutant; Bun-Ya et al., 1991), which is defective in Pi uptake. TaPHT1.1-4D and 1.10-4A were selected to represent the closely related TaPHT1.1/1.2/1.9/1.10 which encoded two types of protein length, 521 amino acids and 525 amino acids. TaPHT1.4-5D was chosen to represent the closely related TaPHT1.3/1.4. The coding regions of the seven selected TaPHT1 genes were separately inserted into the yeast expression vector p112A1NE under the control of the yeast alcohol dehydrogenase promoter. The constructs were separately transformed into a yeast Pi transporter mutant MB192. An empty vector was also transformed to be used as a control (Yp112). We first analyzed the complementation of MB192 by TaPHT1 genes by using dilution based plate assays. All the yeast transformants harboring the candidate TaPHT1 genes grew better than the Yp112 (empty vector control), but poorer than the wild-type in the plates which contained 20, 60, 100, and 140 µM Pi when the yeast cells were diluted to 1/100 OD value (Supplementary Figure S3C). This result indicated that the seven tested TaPHT1 genes could partially restore the growth of MB192 mutant cells. Staining test for acid phosphatase activity also showed that TaPHT1.6-5D and TaPHT1.10-4A partially restore the growth of MB192 mutant (Supplementary Figures S3A,B). We then assessed the kinetic growth of the yeast cells in YNB liquid medium that contained 200 µM Pi (high Pi) and 20 µM Pi (low Pi). The wild-type yeast strain grew much quicker than the Yp112, MB192, and the yeast cells transformed with TaPHT1 genes (Yp112-TaPHT1s), whereas Yp112 and MB192 exhibited a growth defect on both high Pi and low Pi media (Figure 2). All the yeast mutant cells carrying Yp112-TaPHT1s transformants grew faster than Yp112 and MB192 under high Pi and low Pi conditions (Figure 2), suggesting that these selected TaPHT1s had Pi transport activity. ### Responses of TaPHT1 Expression to P Availability Quantitative real-time RT-PCR was used to analyze the responses of TaPHT1 genes to P supply levels at the seedling stage in a hydroponic culture and at the re-greening stage in a field experiment. Primers were designed to amplify the homologous alleles at a particular locus; for example, the relative expression level of TaPHT1.2 might represent that of all three homologous alleles of TaPHT1.2 (TaPHT1.2-4A, -4B, and -4D). In both the hydroponic culture and the field experiment, the expression of TaIPS1.1, a molecular indicator of plant Pi status (Teng et al., 2013), was upregulated by the low P treatment (Supplementary Figures S4A–C), indicating that the plants in the low P treatment in both of the experiments were P-starved. TaPHT1.1/1.9, TaPHT1.2, and TaPHT1.10 were predominantly expressed in roots in both experiments (Figures 3A, 4A), and their expression was dramatically induced by low P treatment in the hydroponic culture (Figure 3A), but not in the field experiment (Figure 4A). Of these four root-specific genes, TaPHT1.10 displayed the highest expression and TaPHT1.1/1.9 the lowest (Figures 3A, 4A). TaPHT1.3/1.4 and TaPHT1.6 were expressed in both roots and shoots, and TaPHT1.6 exhibited stronger expression than TaPHT1.3/1.4 in both experiments (Figures 3B, 4B). These three genes differed in the response 600, Optical density at 600 nm. Data are mean ± SE of three biological replications. to P supply. Compared to high P treatment, low P treatment upregulated TaPHT1.3/1.4 in roots and TaPHT1.6 in shoots in the hydroponic culture (Figure 3B), and upregulated TaPHT1.3/1.4 in roots and shoots and TaPHT1.6 in shoots in the field experiment (Figure 4B). TaPHT1.5, 1.7 and 1.8 were presented at very low expression levels in both roots and shoots in both of the experiments (Figures 3C, 4C). Upregulation by low P treatment was observed for TaPHT1.7 in shoots in the hydroponic culture (Figure 3C), TaPHT1.7 in roots and TaPHT1.8 in roots and shoots in the field experiment (Figure 4C). Since TaPHT1.6 had the most abundant transcripts in shoots among the investigated TaPHT1 genes, we further analyzed the expression of TaPHT1.6 in different aerial parts at the flowering and grain filling stages (14 days after flowering) in the field experiment. The expression of TaPHT1.6 was much higher in leaves than in stems, spikes, and grains, and was higher in aging leaves than in flag leaves (Supplementary Figure S5). Significant upregulation by low P treatment was observed in aging leaves, stems, spikes, and grains (Supplementary Figure S5). ### Relationship of TaPHT1s Expression with P Uptake We measured P uptake of two commercial wheat varieties at stem elongation, flowering, and maturity stages in two consecutive field experiments (2010 experiment and 2011 experiment). Data were collected at the P application rates of 0 kg P ha−<sup>1</sup> (P0), 100 kg P ha−<sup>1</sup> (P100), and 400 kg P ha−<sup>1</sup> (P400). In most cases, the wheat variety KN9204 had higher total P concentration in shoots at stem elongation and flowering and in straws and grains at maturity than the wheat variety SJZ8, except for that of stem elongation in the 2011 experiment (Table 1). Comparison of aerial P accumulation between these two varieties showed that KN9204 absorbed more P than SJZ8 after stem elongation at all the P application rates in both of the field experiments (Figure 5). As the differences in P uptake between KN9204 and SJZ8 were mainly observed at flowering and maturity, we analyzed the TaPHT1s expression at the flowering stage in the 2011 field experiment. The higher expression of TaIPS1.1 at P0 than at P100 and P400 indicated that the wheat plants grown under P0 conditions were P-starved (Supplementary Figure S4D). TaPHT1.1/1.9, 1.2, and 1.10 were expressed more abundantly in the roots of KN9204 than in those of SJZ8 at all the three P rates (Figures 6A–C), whereas SJZ8 had higher expression of TaPHT1.8 in roots at P100 and P400 (Figure 6D) and higher expression of TaPHT1.6 in roots at P0 and P100 (Figure 6E) and in shoots at P0 than KN9204 (Figure 6F). The paired t-test showed that the mean values across the three P application rates for P uptake after stem elongation in 2010 and 2011 field experiments and the expression of TaPHT1.1/1.9 and 1.10 at flowering in 2011 field experiment were significantly higher in KN9204 than in SJZ8 (Supplementary Table S4). We further analyzed the correlations between gene expression at flowering and P uptake after stem elongation (difference between stem elongation and maturity). P uptake after stem elongation showed a positive correlation with the expression of TaPHT1.1/1.9 (Figure 7A), TaPHT1.2 (Figure 7B), and TaPHT1.10 in roots (Figure 7C), but a negative correlation with the expression of TaPHT1.8 in roots (Figure 6D) and TaPHT1.6 in roots and shoots (Figures 6E,F). ### DISCUSSION We identified a total of 36 TaPHT1 genes named from TaPHT1.1 to TaPHT1.14 in wheat. Of the 32 PHT1 genes with chromosome location information, 12, 11, and 9 were from the A, B, and D genomes (Supplementary Table S3), respectively. In order to evaluate the PHT1 number in wheat, we also identified 13 PHT1 genes in Triticum urartu and 14 PHT1 genes in Aegilops tauschii, and these PHT1 genes were named from PHT1.1 to PHT1.16 (Supplementary Table S3). We did not find the wheat Gene IDs which are orthologous to PHT1.15 and PHT1.16 of Triticum urartu and Aegilops tauschii (Figure 1), but several scaffolds from the short arms of group 2 chromosomes in wheat contained PHT1.15 and PHT1.6 like fragments which were not annotated yet. Although we identified a PHT1.14 gene (TaPHT1.14-U) in wheat, there were three closely related PHT1.14 genes (AEGtaPHT1.14-1, 1.14-2, and 1.14-3) in Aegilops tauschii (Figure 1). Further detailed analysis of the Chinese spring genome sequence found that the forward orientation of seven fragments showed high similarity with TaPHT1.14- U in the scaffold TGACv1\_scaffold\_642582\_U (Supplementary Figure S6). The second, fourth, fifth, and sixth fragments showed similarity only with the 3<sup>0</sup> -end of TaPHT1.14-U (Supplementary Figure S6). However, the first fragment showed 96–98% identity with 1–1604 bp of the 1656 bp coding region in TaPHT1.14-U (the third fragment 3, Supplementary Figure S6), whereas the seventh fragment located in the last 550 bp of the scaffold showed 99% identity with 1–550 bp of the coding region in TaPHT1.14-U (Supplementary Figure S6). As such, TGACv1\_scaffold\_642582\_U may contain three closely related PHT1.14 genes. Taking the information together, there may have as many as 16 (if with one PHT1.14 gene)-18 (if with three PHT1.14 genes) PHT1 genes in each of the three subgenomes in wheat. The cloned 10 TaPHT1 promoters contained the putative Pi-starvation response regulator PHR1 binding cis-element P1BS Different letters (a and b) indicate significant difference between SJZ8 and KN9204 at p < 0.05 level. and the WRKY transcription factor binding element W-Box (Supplementary Figure S2), indicating that TaPHT1s may be regulated by PHR1 and WRKY transcription regulatory factors. In fact, our previous study has documented that TaPHR1 can form homodimers to activate TaPHT1.10-U expression in vitro (Wang et al., 2013). It has been reported that PHR1 regulates Pi starvation-inducible genes by binding as a dimer to the cis-element P1BS in the promoter region of its downstream gene (Rubio et al., 2001) and the majority of Pi starvation-inducible genes contain the P1BS element (Muller et al., 2007; Nilsson et al., 2010). As such, the P1BS elements in the promoters might contribute to the observed upregulation of TaPHT1 genes by low P treatment (Figure 3). Several WRKY transcription factors have been found to bind to the W-box to regulate the expression of Pi-starvation response genes in Arabidopsis (Devaiah et al., 2007; Chen et al., 2009; Wang et al., 2014). Whether WRKY transcription factors involved in regulating the response of TaPHT1 genes to P-deficiency is needed to be studied in the future. The seven tested TaPHT1 genes showed Pi-transport activity in yeast cells grown under low Pi and high Pi conditions (Figure 2). The genes from the same branch of the phylogenetic tree shared similar tissue-specific expression and response to P-deficiency (Figures 3, 4). The expression of TaPHT1.1/1.2/1.9/1.10 in Branch V was root-specific and upregulated by low P treatment in the hydroponic culture (Figure 3A), but their upregulations by low P treatment was abolished in the field experiments (Figures 4A, 6A–C). These abolished upregulations by low P treatment were possible due to the increased AM colonization in roots under P deficiency (Teng et al., 2013), as AM colonization has been found to inhibit the response of HvPHT1.1 and HvPHT1.2 to P-deficiency in roots of barley (Glassop et al., 2005). In Branch V, HvPHT1.1 has been found to encode high-affinity transporters of Pi (Preuss et al., 2011). Taking information together, TaPHT1 transporters in Branch V may function as high-affinity Pi transporters mediating Pi acquisition from soils. TaPHT1.6 in Branch III and TaPHT1.3/1.4 in Branch IV were expressed in both roots and shoots and were upregulated by low P treatment in the hydroponic culture and in the field experiment (Figures 3B, 4B). In aerial parts, TaPHT1.6 was expressed in stems, leaves, spikes, and grains (Supplementary Figure S5). HvPHT1.6 in Branch III was expressed in both roots and shoots (Rae et al., 2003). OsPHT1.8 in Branch IV was expressed in various tissue organs from roots to seeds and plays an important role in Pi homeostasis and P redistribution from source to sink organs (Jia et al., 2011; Li et al., 2015). These results indicate that PHT1s in Branches III and IV may mediate Pi remobilization in whole plant. However, they may have diverse affinities for Pi, as OsPHT1.8 has been shown high-affinity for Pi (Jia et al., 2011), and HvPHT1.6 low-affinity for Pi (Rae et al., 2003). TaPHT1.5 in Branch IV and TaPHT1.7 in Branch III were expressed at very low levels in both of the hydroponic culture and field experiment (Figures 3C, 4C). The reported AM fungi inducible PHT1s were grouped into Branch VI (Figure 1), including TaPHT1.8 and TaPHT1.11 from wheat, HvPHT1.8 and HvPHT1.11 from barley, ZmPHT1.6 from maize (Glassop et al., 2005; Sisaphaithong et al., 2012), and OsPHT1.11 from rice (Paszkowski et al., 2002). Here, we found that TaPHT1.8 was upregulated by low P treatment in the field experiments (Figures 4C, 6D), and P < 0.01, respectively. but not by low P treatment in the hydroponic culture (Figure 3C). Since we observed that low P treatment increased AM colonization rate in roots of KN9204 compared to high P treatment in field experiments (Teng et al., 2013), the upregulation of TaPHT1.8 by low P treatment in the field experiments might reflect the fact that TaPHT1.8 was exclusively induced by AM fungi (Glassop et al., 2005). Previous studies state that transgenic modifying expression of PHT1 genes altered P uptake and re-distribution in wheat (Liu et al., 2013) and rice (Ai et al., 2009; Jia et al., 2011; Yan et al., 2014). The transcript abundance of PHT1 genes has been shown to relate with P uptake in wheat (Aziz et al., 2014) and P utilization efficiency in barley (Huang et al., 2011) under controlled conditions. These results indicate that mRNA levels of PHT1 genes affect the capacities of P uptake and remobilization. Our current on-farm field-scale study showed that the expression of TaPHT1.1/1.2/1.9/1.10 correlated with the differences in P uptake between different wheat varieties. The positive correlations between P uptake after stem elongation and the expression levels of TaPHT1.1/1.2/1.9/1.10 at the flowering stage (Figures 7A–C) might result from two factors: P supply level and wheat variety. Firstly, both P uptake and expression of these TaPHT1 genes increased with P application rate (Figures 5, 6A–C). Secondly, KN9204 had higher P uptake after stem elongation and the relative expression levels of TaPHT1.1/1.2/1.9/1.10 at the flowering stage than SJZ8 at a given P application rate (Figures 5, 6A–C). In contrast to the positive correlations between the expression of TaPHT1.1/1.2/1.9/1.10 and P uptake after stem elongation, the expression of TaPHT1.8 and TaPHT1.6 negatively correlated with P uptake after stem elongation (Figures 7D–F). The negative correlation between TaPHT1.8 expression and P uptake after stem elongation resulted from the decreased TaPHT1.8 expression with P application rate and lower TaPHT1.8 expression in roots of KN9204 compared to that of SJZ8 at P100 and P400 (Figure 7D). However, this negative correlation did not support that AM colonization inhibited P uptake, as we did not analyze the expression of TaPHT1.11 yet. It has been reported that TaPHT1.11-A1, -B1, and -D1 were AM-inducible and were expressed at much higher level than TaPHT1.8 (Sisaphaithong et al., 2012). The negative correlation between TaPHT1.6 expression and P uptake after stem elongation mainly resulted from the decreased TaPHT1.6 expression with P application rate (Figures 7E,F), as KN9204 and SJZ8 had similar expression levels of TaPHT1.6 in roots at P400, and in shoots at P0, P100 and P400 (Figures 6E,F). Although TaPHT1.6 may mediate P redistribution, the similar expression levels of TaPHT1.6 in shoots at flowering did not explain the differences in grain P concentration between these two varieties (Table 1). This was possibly because that the transport of P to grains occurs during grain filling. As such, further research is needed to investigate the expression of TaPHT1 genes including TaPHT1.6 ### REFERENCES Ai, P. H., Sun, S. B., Zhao, J. N., Fan, X. R., Xin, W. J., Guo, Q., et al. (2009). Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions during grain filling, the research may identify the TaPHT1 genes which contribute to the differences in grain P concentration between different wheat varieties. In summary, the hexaploid wheat has many more PHT1 genes than the diploid cereal crops such as barley and rice. Although we performed genome-wide analysis of PHT1 genes, we did not isolate all the PHT1 genes in wheat. The on-going wheat genome sequencing project will help us to understand the complexity of the Pi transport system in wheat. Although there were a large number of PHT1 genes in wheat, the TaPHT1 transporters from a given branch of the phylogenetic tree shared high similarities in sequences, expression locations, and responses to P-availability, this finding will help us to predict the roles of TaPHT1 genes in mediating Pi uptake and re-distribution. Our research also provided useful cues to understand the influences of PHT1 genes on the genotypic differences in P uptake. Further studies on mechanisms underlying the genotypic differences in PHT1 expression will facilitate the breeding of wheat varieties with improve P use efficiency. ### AUTHOR CONTRIBUTIONS Y-PT, WT, and Y-YZ designed this study; Y-YZ screened BAC clones, WT and Y-YZ analyzed PHT1 sequences; Y-YZ and WT assayed expression and function of PHT1 genes; all authors carried out the field experiments; WT and Y-YZ wrote the manuscript under the supervision of Y-PT. All authors have read and approved this manuscript. ### FUNDING This research was supported by the National Key Research and Development Program of China from Ministry of Science and Technology of China (2016YFD0100706) and the National Transgenic Key Project from the Ministry of Agriculture of China (2016ZX08002-005). ### ACKNOWLEDGMENT The yeast mutant MB192 and vector p112A1NE were kindly provided by Prof. Shubin Sun from Nanjing Agricultural University, Nanjing, China. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00543/ full#supplementary-material and kinetic properties in uptake and translocation. Plant J. 57, 798–809. doi: 10.1111/j.1365-313X.2008.03726.x Aziz, T., Finnegan, P. M., Lambers, H., and Jost, R. (2014). Organ-specific phosphorus-allocation patterns and transcript profiles linked to phosphorus efficiency in two contrasting wheat genotypes. Plant Cell Environ. 37, 943–960. doi: 10.1111/pce.12210 Conflict of Interest Statement:* The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Teng, Zhao, Zhao, He, Ma, Deng, Chen and Tong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.	doab	2025-04-07T03:56:58.883365	18-11-2021 17:11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005998e1-f40a-4c86-b95d-ac77fff26206", "url": "https://www.frontiersin.org/research-topics/5889/improving-the-nutritional-content-and-quality-of-crops-promises-achievements-and-future-challenges", "author": "", "title": "Improving the Nutritional Content and Quality of Crops: Promises, Achievements, and Future Challenges", "publisher": "Frontiers Media SA", "isbn": "9782889630189", "section_idx": 0 }
005f4cc4-de17-43d3-8915-84cd53654c1d.0	Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms • Stefano Perna, Francesco Soldovieri and Moeness Amin	doab	2025-04-07T03:56:58.977107	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 0 }
005f4cc4-de17-43d3-8915-84cd53654c1d.1	Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms	doab	2025-04-07T03:56:58.977210	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 1 }
005f4cc4-de17-43d3-8915-84cd53654c1d.2	Edited by Printed Edition of the Special Issue Published in Remote Sensing Stefano Perna, Francesco Soldovieri and Moeness Amin www.mdpi.com/journal/remotesensing	doab	2025-04-07T03:56:58.977245	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 2 }
005f4cc4-de17-43d3-8915-84cd53654c1d.3	Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms ## Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms Special Issue Editors Stefano Perna Francesco Soldovieri Moeness Amin MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Remote Sensing (ISSN 2072-4292) from 2018 to 2020 (available at: https://www.mdpi.com/journal/ remotesensing/special issues/radarimaging). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number, Page Range. ISBN 978-3-03936-469-5 (Pbk) ISBN 978-3-03936-470-1 (PDF) c 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.	doab	2025-04-07T03:56:58.977278	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 3 }
005f4cc4-de17-43d3-8915-84cd53654c1d.4	Contents ### Chunhui Lin, Shiyang Tang, Linrang Zhang and Ping Guo ### About the Special Issue Editors Stefano Perna completed his Laurea Degree (summa cum laude) in Telecommunication Engineering and his PhD in Electronic and Telecommunication Engineering, both at the Universita degli Studi ` di Napoli "Federico II", Naples, Italy, in 2001 and 2006, respectively. Since 2006, he has been with the Department of Engineering (DI) at the Universita degli Studi di Napoli "Parthenope", Naples, ` where he is currently a researcher in electromagnetics and teaches the courses "Antennas" and "Electromagnetics". He is currently also an adjunct researcher at IREA-CNR, Naples. Since 2015, he has collaborated with the Argentinian National Council of Technical and Scientific Research (CONICET) on activities relevant to the focusing and processing of SAR data acquired by the airborne SARAT system. In 2016, he was a visiting professor at the Departamento de Teor´ıa de la Senal y Comunicaciones of the Universitat Polit ˜ ecnica de Catalunya (UPC), Barcelona, Spain. He is ` an IEEE senior member. His main research interests are in the field of microwave remote sensing and electromagnetics: airborne SAR data modelling and processing, airborne differential SAR interferometry, modelling of electromagnetic scattering from natural surfaces, synthesis of antenna arrays; antenna characterization and measurement in anechoic and reverberating chambers. He is the co-author of about 100 papers published in international scientific journals or proceedings of international conferences in the field of electromagnetics and remote sensing. Francesco Soldovieri is a research director at Institute for Electromagnetic Sensing of the Environment of CNR. He was General Chair of the International Workshop on Advanced Ground Penetrating Radar 2007 and General Co-Chair of the Ground Penetrating Radar Conference 2010. He was a member of the Editorial Board of IEEE-GRSL and now of IEEE-TCI and IEEE-TGRS, Remote Sensing (MDPI). He is Editor in Chief of HERITAGE, a MDPI journal devoted to cultural and natural heritage. He was the scientific coordinator of the FP7 projects ISTIMES and AMISS and the technical manager of the H2020 Project HERACLES. He was the President of the Division on Geosciences Instrumentation and Data Systems of the European Geosciences Union. His research interests include radar imaging, data processing for GPR, indoor surveillance, through-wall imaging, passive radars, integration of geophysical data, and radars for planetary exploration. He is the co-author of about 240 papers in national and international journals, and more than 350 conference proceedings. Moeness Amin completed his BSc degree at the Faculty of Engineering, Cairo University in 1976, and his MSc at the University of Petroleum and Minerals in 1980. He then completed a PhD at the University of Colorado, Boulder, in 1984; all degrees are in Electrical Engineering. Since 1985, he has been with the Faculty of the Department of Electrical and Computer Engineering, Villanova University, Villanova, PA, USA, where he became the Director of the Center for Advanced Communications, College of Engineering, in 2002. Dr. Amin is a Fellow of the Institute of Electrical and Electronics Engineers (IEEE); Fellow of the International Society of Optical Engineering (SPIE); Fellow of the Institute of Engineering and Technology (IET); and a Fellow of the European Association for Signal Processing (EURASIP). He is the recipient of: the 2017 Fulbright Distinguished Chair in Advanced Science and Technology; the 2016 Alexander von Humboldt Research Award; the 2016 IET Achievement Medal; the 2014 IEEE Signal Processing Society Technical Achievement Award; the 2009 Technical Achievement Award from the European Association for Signal Processing; the 2015 IEEE Aerospace and Electronic Systems Society Warren D White Award for Excellence in Radar Engineering. He is also the recipient of the IEEE Third Millennium Medal. Dr. Amin has over 800 journal and conference publications in signal processing theory and applications, covering the areas of wireless communications, radar, sonar, satellite navigations, ultrasound, healthcare, and RFID. He has co-authored 23 book chapters and is the editor of three books titled, Through the Wall Radar Imaging, Compressive Sensing for Urban Radar, and Radar for Indoor Monitoring, all published by CRC Press, in 2011, 2014, 2017, respectively. ### Editorial Editorial for Special Issue "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms" ### *Stefano Perna 1,2,\, Francesco Soldovieri 2 and Moeness Amin 3 Received: 15 April 2020; Accepted: 15 April 2020; Published: 17 April 2020 Abstract: Microwave radar imaging plays a key role in several civilian and defense applications, such as security, surveillance, diagnostics and monitoring in civil engineering and cultural heritage, environment observation, with particular emphasis on disasters and crisis management, where it is required to remotely sense the area of interest in a timely, safe and e ffective way. To address these constraints, a technological opportunity is o ffered by radar systems mounted onboard smart and flexible platforms, such as ground-based ones, airplanes, helicopters, drones, unmanned aerial and ground vehicles (UAV and UGV). For this reason, radar imaging based on data collected by such platforms is gaining interest in the remote sensing community. However, a full exploitation of smart and flexible radar systems requires the development and use of image formation techniques and reconstruction approaches able to exploit and properly deal with non-conventional data acquisition configurations. The other main issue is related to the need to operate in challenging environments, and still deliver high target detection, localization and tracking. These environments include through the wall imaging, rugged terrain and rough surface/subsurface. In these cases, one seeks mitigation of the adverse e ffects of clutter and multipath via the implementation of e ffective signal processing strategies and electromagnetic modeling. This Special Issue (SI) is aimed at providing an overview of recent scientific and technological advances in the field of radar imaging from smart and flexible platforms, in terms of hardware, modeling and data processing. The contributions of the SI can be generally classified into two groups. The papers belonging to the first group [1–6] provide the description of the capabilities of newborn imaging radar systems designed to operate in challenging scenarios [1] or using smart and flexible aerial platforms, such as small airplanes [2], drones [3–5] or helicopters [6]. Overall, these contributions provide an interesting survey of the potential of lightweight and compact imaging radar sensors. The described systems cover a very wide range of the microwave spectrum, including the VHF band, up to the X-band. The papers under this group [5] provide a good survey of the radar hardware as well as the corresponding processing chain applied to the acquired data. The contributions belonging to the second group [7–10] are focused on the description of novel data processing techniques aimed at achieving accurate radar imaging under complex acquisition geometries, such as in the case of airborne Synthetic Aperture Radar (SAR) [6–8], or in challenging scenarios, as in the case of Forward-Looking Ground-Penetrating Radar (FL-GPR) [9] or Lunar Penetrating Radar (LPR) [10]. As for the papers belonging to the first group, in [1], a newborn Ultra Wideband (UWB) Multiple-Input Multiple-Output (MIMO) radar system exploiting the Stepped-Frequency Continuous-Wave (SFCW) technology to detect human targets beyond the obstacle, is presented. More specifically, the design, as well as manufacturing processes leading to the realization of the overall radar system, which also includes a novel miniaturized Vivaldi antenna with 0.5–2.5 GHz bandwidth, are described. The radar system is successfully used for through-wall imaging applications by exploiting a data-processing algorithm based on the Cross-Correlation Time Domain Back Projection (CC-TDBP) technique. In [2–4], two newborn SAR systems mounted onboard aerial platforms are presented. In particular, in [2], the imaging and topographic capabilities of a novel Italian airborne X-band SAR system, named AXIS, are discussed. The system is based on the Frequency-Modulated Continuous-Wave (FMCW) technology and is equipped with a single-pass interferometric layout. In this work, the description of the developed radar system is given along with a quantitative assessment of the quality of the SLC (Single Look Complex) SAR images and the interferometric products achievable through the system. In [3,4], a novel Brazilian drone-borne SAR system operating in three di fferent frequency bands, namely the C-, L- and P-band, is presented. The system is capable of exploiting a single-pass interferometric configuration at C-band, and full-polarimetric configurations at the L- and P-band. In [3], the description of the system and a quantitative assessment of the results achieved by applying the Di fferential SAR Interferometry (DInSAR) technique to the L-band data is presented. The work in [4] is focused on an interesting precision farming application scenario enabled by the exploitation of the drone-borne SAR system. More specifically, a novel methodology for obtaining growth deficit maps with an accuracy down to 5 cm and a spatial resolution of 1 m is presented. The proposed methodology is based on the DInSAR technique. Another light and compact imaging radar system mounted onboard a small Multicopter-Unmanned Aerial Vehicle (M-UAV) is presented in [5]. In this case, the radar operates with 1.7 GHz bandwidth centered at 3.95 GHz, and the flight positions are obtained through the Carrier-Phase Di fferential GPS (CDGPS) technique. In particular, the work describes the overall radar imaging system in terms of both hardware devices and data processing strategy. The system is validated by collecting and processing a dataset through a single flight track to provide focused images of on surface targets. In [6], a helicopter-borne integrated Sounder/SAR system operating in the UHF and VHF frequency bands is described. More specifically, the Sounder operates at 165 MHz, whereas the full-polarimetric SAR could operate either at 450 MHz or at 860 MHz. The system is developed under the auspices of a contract between the Italian Space Agency (ASI) and di fferent private and public Italian Research Institutes and Universities. In this work, the first results relevant to a set of Sounder and SAR data, acquired during a campaign conducted in 2018 over a desert area in Erfoud, Morocco, are presented. As for the papers belonging to the second group, they address the processing of three kinds of imaging radar data, namely, airborne SAR [7,8], FL-GPR [9] and LPR [10] data. For airborne SAR processing, exploitation of small and flexible aerial platforms to mount the radar systems makes the issues related to motion errors (that is, the attitude and position instabilities of the platform during the acquisition) coupled to the topographic variations of the observed scene even more critical; therefore, ad-hoc data processing strategies capable to properly account for these problems are needed. In [7], the spatial variations induced on airborne SAR images by the motion errors are decomposed into three main parts: range, azimuth and cross-coupling terms. The cross-coupling variations are then corrected by means of a polynomial phase filter, whereas the range and azimuth terms are removed through Stolt mapping. In [8], an extended back-projection approach is proposed to take into account the topography variations during the airborne SAR image formation process. In particular, the algorithm applies a time–frequency rotation operation to pursue high accuracy, while reducing the computational burden, typically required by standard back-projection algorithms operating entirely in the time-domain. The FL-GPR allows fast scanning of large areas for real-time target detection, unlike its ground-coupled or near-ground down-looking GPR (DL-GPR) counterparts. This capability, however, comes at the expense of energy backscattered from the illuminated targets and limited image spatial resolution. Furthermore, the rough ground surface generates clutter that may obscure the buried targets, rendering target detection very challenging. In this respect, the work in [9] presents an enhanced imaging procedure for the suppression of the rough surface clutter arising in FL-GPR applications. The procedure is based on a matched filtering formulation of microwave tomographic imaging enhanced by a coherence factor (CF) scheme for clutter suppression. The work in [10] is framed in the context of the planetary exploration and deals with the Lunar Penetrating Radar mounted onboard the Yutu lunar rover to detect the lunar regolith and the shallower subsurface geologic structures of the Moon. In particular, it is aimed at improving the capability of identifying response signals caused by discrete reflectors (such as meteorites, basalt and debris) beneath the lunar surface. To this end, a compressive sensing (CS)-based approach is proposed to estimate the amplitudes and time delays of the radar signals from LPR data. In conclusion, this informative Special Issue would not have been possible without the hard work of all authors and reviewers. We also would like to extend our sincere appreciation to the Editorial O ffice of Remote Sensing for their professional and excellent managemen<sup>t</sup> work. Author Contributions: The authors contribute equally to write this Editorial. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest:** The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.977372	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 4 }
005f4cc4-de17-43d3-8915-84cd53654c1d.15	Abbreviations The following abbreviations are used in this manuscript:	doab	2025-04-07T03:56:58.978476	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 15 }
005f4cc4-de17-43d3-8915-84cd53654c1d.17	[CrossRef] c 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com Remote Sensing Editorial Office E-mail: [email protected] www.mdpi.com/journal/remotesensing MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel: +41 61 683 77 34 Fax: +41 61 302 89 18	doab	2025-04-07T03:56:58.978514	1-5-2021 17:31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "005f4cc4-de17-43d3-8915-84cd53654c1d", "url": "https://mdpi.com/books/pdfview/book/2641", "author": "", "title": "Radar Imaging in Challenging Scenarios from Smart and Flexible Platforms", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039364695", "section_idx": 17 }
0067e0a8-b387-4d2e-a700-d437204e9c1c.0	# Integrative Multi-Omics in Biomedical Research Edited by Michelle Hill and Christopher Gerner Printed Edition of the Special Issue Published in Biomolecules www.mdpi.com/journal/biomolecules	doab	2025-04-07T03:56:58.982328	11-1-2022 14:51	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0067e0a8-b387-4d2e-a700-d437204e9c1c", "url": "https://mdpi.com/books/pdfview/book/4705", "author": "", "title": "Integrative Multi-Omics in Biomedical Research", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036525822", "section_idx": 0 }
0067e0a8-b387-4d2e-a700-d437204e9c1c.1	Integrative Multi-Omics in Biomedical Research	doab	2025-04-07T03:56:58.982405	11-1-2022 14:51	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0067e0a8-b387-4d2e-a700-d437204e9c1c", "url": "https://mdpi.com/books/pdfview/book/4705", "author": "", "title": "Integrative Multi-Omics in Biomedical Research", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036525822", "section_idx": 1 }
0067e0a8-b387-4d2e-a700-d437204e9c1c.2	Integrative Multi-Omics in Biomedical Research Editors Michelle Hill Christopher Gerner MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Michelle Hill Cell Molecular Biology QIMR Berghofer Medical Research Institute Australia Christopher Gerner Department of Analytical Chemistry University of Vienna Austria Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Biomolecules (ISSN 2218-273X) (available at: http://www.mdpi.com). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number, Page Range. ISBN 978-3-0365-2582-2 (Hbk) ISBN 978-3-0365-2583-9 (PDF) © 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. ## Contents #### Timothy R. Howard and Ileana M. Cristea Interrogating Host Antiviral Environments Driven by Nuclear DNA Sensing: A Multiomic Perspective Reprinted from: Biomolecules 2020, 10, 1591, doi:10.3390/biom10121591 ............... 145 ## About the Editors Michelle Hill obtained her PhD at the University of Queensland, Brisbane, Australia. After postdoctoral fellowships with Brian Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) and Seamus Martin (Trinity College Dublin, Dublin, Ireland), she worked as Research Officer with Robert Parton and John Hancock at Institute for Molecular Bioscience, The University of Queensland. In 2009, Michelle established the Cancer Proteomics Group at the University of Queensland Diamantina Institute at the Princess Alexandra Hospital campus, which relocated to QIMR Berghofer Medical Research Institute as Precision and Systems Biomedicine Laboratory. Her research aims to improve modifiable health outcomes by developing better diagnostics and integrative study of cell membrane disturbance that lead to diseases such as cancers, chronic diseases and infection. The program focus on early disease detection and non-drug prevention approaches, to provide tools for actively maintaining health. Their research combines cell biology and biochemistry knowledge with cutting edge multi-omics technologies and computational analyses. Christopher Gerner studied biochemistry at the University of Vienna. After his post-doc in the labs of Rolf Schulte-Hermann, University of Vienna and Seamus Martin, Trinity College Dublin, he became Associate Professor at the Medical University of Vienna in 2003, then heading a clinical proteomics laboratory. 2012 he became full professor for bioanalytics at the University of Vienna. He is now heading the Joint Metabolome Facility, focusing on post-genomic analysis of clinical samples. Thus, he is characterizing patho-mechanisms mainly governed by biochemical processes such as hypoxia, oxidative stress or metabolic shortcomings. In his lab, several methods supporting analysis of humans in vivo (e.g., metabolomics based on finger sweat analysis) and the analysis of human or animal model-derived materials with regard to proteins, lipids and metabolites are well established. Corroborated by phospho-proteomics analyses focusing on short-term effects, this toolbox proves highly versatile for the investigation of drug effects and disease mechanisms. ## Editorial Integrative Multi-Omics in Biomedical Research *Michelle M. Hill 1,2,\ and Christopher Gerner <sup>3</sup> Genome technologies have revolutionized biomedicine, but the complexity of biological systems cannot be explained by genomics alone. Advances in sequencing and mass spectrometry technologies coupled with methodological and computational innovations are essential in driving multidimensional omics applications. This Special Issue covers the latest methods and novel findings from integrative analysis of multiple omics datasets to address diverse questions in biology and pathology. The scene is set with a review article by Lancaster et al. [1], which introduces six players (genome, epigenome, transcriptome, metagenome, proteome and metabolome) that use two different technologies (sequencing and mass spectrometry). After characterizing individual omics data and analytical approaches, considerations for multi-omic study design and data integration methods are discussed. The contributed research papers span a broad range of studies from clinical cohorts and mouse models to cell-based investigations, thus illustrating the diverse applications of multi-omics. Two papers applied multi-omics to investigate physiological interventions. Odenkirk et al. [2] compared the blood lipidome and metabolome in two cohorts of patients undergoing exercise and planned myocardial infarction, respectively, to gain insight on the metabolic pathways underlying the disease and its prevention. Molendijk et al. [3] applied lipidomic and metagenomic profiling in a dietary model of gastro-esophageal reflux disease and associated esophageal pathology in mice, revealing increased microbiome diversity and a lipidomics signature associated with esophageal inflammation and metaplasia. Five papers applied multi-omics to diverse cell models, with a study by Niederstaetter et al. [4] highlighting the variability and influence of fetal calf serum (used in culture media)-contained eicosanoids on cellular function, evaluated via proteomics and lipidomics. Neuditschko et al. [5] investigated endometrial pain mechanisms by applying proteomics, metabolomics and eicosanoid profiling to cells derived from endometriotic lesions. Gillen et al. [6] applied metabolic measurements with secretome profiling to assess the impact of endotoxin (LPS) on macrophages, while Novikova et al. [7] combined transcriptome and proteomic profiling to investigate granulocyte differentiation and discovered HIC1, CEBPB, LYN and PARP1 as potential therapeutic targets in acute myeloid leukemia. Finally, the paper by Kim et al. [8] illustrates the standardized application of combining drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) to facilitate target protein identification for other existing natural therapeutic compounds. To wrap up this Special Issue, the comprehensive review article by Howard and Cristea [9] highlights the role of integrative multi-omics in deciphering system-level mechanisms of DNA sensing during viral infections. Following viral infection, protein–protein interactome and protein post-translational modifications drive the remodeling of the cellular transcriptome, proteome and secretome; hence, multi-omic investigations should also include interactome and modification analyses such as phosphoproteome. Citation:** Hill, M.M.; Gerner, C. Integrative Multi-Omics in Biomedical Research. Biomolecules 2021, 11, 1527. https://doi.org/ 10.3390/biom11101527 Received: 6 October 2021 Accepted: 8 October 2021 Published: 16 October 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). In conclusion, multi-omic investigation has become a central technique for deciphering complex biological systems. Continued innovations in technologies, methodologies and applications will enable and support further expansion and integration of multi-omics in future biomedical research. Conflicts of Interest: The authors declare no conflict of interest. #### References ## Review A Customizable Analysis Flow in Integrative Multi-Omics ### *Samuel M. Lancaster 1,2,\, Akshay Sanghi 1,2,\, Si Wu 1,2,\ and Michael P. Snyder 1,2,\* Received: 30 September 2020; Accepted: 23 November 2020; Published: 27 November 2020 Abstract: The number of researchers using multi-omics is growing. Though still expensive, every year it is cheaper to perform multi-omic studies, often exponentially so. In addition to its increasing accessibility, multi-omics reveals a view of systems biology to an unprecedented depth. Thus, multi-omics can be used to answer a broad range of biological questions in finer resolution than previous methods. We used six omic measurements—four nucleic acid (i.e., genomic, epigenomic, transcriptomics, and metagenomic) and two mass spectrometry (proteomics and metabolomics) based—to highlight an analysis workflow on this type of data, which is often vast. This workflow is not exhaustive of all the omic measurements or analysis methods, but it will provide an experienced or even a novice multi-omic researcher with the tools necessary to analyze their data. This review begins with analyzing a single ome and study design, and then synthesizes best practices in data integration techniques that include machine learning. Furthermore, we delineate methods to validate findings from multi-omic integration. Ultimately, multi-omic integration offers a window into the complexity of molecular interactions and a comprehensive view of systems biology. Keywords: multi-omics; multi-omics analysis; study design; bioinformatics; machine learning; analysis flow #### 1. Introduction Omics measurements are unbiased samples of molecules from a biological specimen. The genome was the first ome studied [1,2], and subsequent omes followed, building off DNA sequencing technology. Transcriptomics sequences the RNA content in cells, and metagenomics sequences all the genetic material from a group of organisms, usually microbial populations. Chromatin accessibility measurements select for sections of DNA to sequence that are differentially bound by chromatin—believed to affect transcription. Omic measurements are not limited to nucleic acid sequencing. The most common omics methods orthologous to nucleotide sequencing involve mass spectrometry (MS). These include proteomics, metabolomics, and lipidomics, which are all vitally important and account for innumerable actionable discoveries. There are many other omic measurements, which all work together to improve understanding of systems biology. Understanding each of these omes is vitally important and integrating them provides a more comprehensive picture of biology. For example, to understand the biochemical effects of changes in transcription, one must understand the metabolome and proteome as well. However, with the different natures of omic measurements, and the fact that they are best modeled by different statistical distributions, integrating this vast information in these distinct biological layers is challenging to non-experts. Using these omic measurements as examples, we will highlight potential integration methods that will reveal trends in multi-omics data. #### 2. Analysis of Single Omics Prior to Integration** Each of these omic methods is analyzed differently, with similar analyses shared between the more similar methods. One cannot discuss multi-omic integration without first having a shared understanding of how to analyze the individual omic measurements. #### 2.1. Genome Analysis The genome is the core ome, and it codes for the basic information that inevitably is pushed into the other omes. For example, the transcriptome is aligned with the genome. This task is complicated because of the numerous mRNA isoforms, and the non-normal distribution of reads, which can be modeled using a negative binomial distribution [3]. After alignment and normalization, the read depth is used as a measurement of expression, reviewed below. Similarly, in the metagenome data, reads are aligned with the set of known microbiome data and read depth is assumed to be an abundance of each microorganism [4]. Chromatin accessibility measurements, such as the assay for transposase-accessible chromatin using sequencing (ATACseq), follow a similar principle. In this case read depth is a measure for how open the chromatin is. Most genomes are sequenced on an Illumina platform, generating short reads. First, the quality of these reads must be determined, which informs one how well the sequencing was performed. Generally speaking a PHRED score of 30 is used as a threshold for keeping a read, although it may be altered depending on the needs of a study [5]. These scores are saved in FASTQ files as one of the four rows for each read, and they may be pulled out using several different programs. Another main sequencing type, long read sequencing, usually allows for the retrieval of much longer (>10,000 bp) sequencing reads (e.g., PacBio) and may be used to better capture repetitive regions or insertions or deletions, but it is often more expensive per base. The reads that pass quality controls must be aligned with a known genome. For organisms without assembled reference genomes, which are increasingly rare, such reads must first be assembled into a genome with large contiguous chunks of DNA, or contigs (reviewed in [6]). Alignment tools such as BWA and Bowtie allow alignment of reads with a given number of mismatches, because no genome will be identical to the reference genome [7,8]. These alignments generate a sequence alignment map (SAM) file and their more compressed binary format BAM file [9]. From these files, variants between the sequenced genome and referenced genome can be determined using Samtools or other software and saved as a variance call format (VCF) file [10]. These may be DNA insertions, deletions, or nucleotide variations. From these files, biologically relevant genetic differences, for example, those that affect protein translations, may be determined. In some cases, single nucleotide polymophisms (SNPs) can be associated with known phenotypes or may even be proved causative for a disease. #### 2.2. Epigenomic Analysis Epigenomic analysis aims to understand the functional context of the genome. For example, an animal has multiple organs with the same genome, but the genes expressed vary between organs depending on the tissue's epigenetic state. The genome is contained within a larger chromatin context that regulates which genes have access to transcriptional machinery and which are insulated from active machinery. Various technologies have been developed to profile the epigenetic landscape, and particularly in the last decade, next-generation technologies have been applied to comprehensively map the epigenetic patterns in mammalian species [11,12]. One of the newest technologies in epigenetic analysis is assay transposase-accessible chromatin sequencing (ATAC-seq) [13]. The benefits of the ATAC-seq are (1) it provides direct evidence of genomic positions of nucleosome-depleted chromatin, which are permissible to transcriptional machinery binding, and (2) the assay only requires 10,000–50,000 cells as input, so it is particularly useful for animal tissue and limited specimens [14]. Similarly to whole-genome sequencing, ATAC-seq data are generated on the Illumina platform, giving high resolution information of open chromatin regions throughout the entire genome. After alignment with the same genome aligners, such as Bowtie, a critical step is filtering out low-quality and insignificant reads. This especially entails removing the high fraction of mitochondrial reads, which because of their high degree of accessibility are preferentially ligated with sequencing adapters. The sequencing reads are then analyzed for their pile-ups in peaks. The general purpose of peak calling is to find regions (on the order of hundreds of base pairs) that have significantly more reads piled up compared to the background reads across the genome [15]. ATAC-seq peaks represent the functional output of the data, and peaks are used in several types of analyses [16]. One very interesting analysis is transcription factor footprinting, which predicts which transcription factors are actively bound to chromatin and where the transcription factors activate transcription, giving insights into the regulatory pathways that affect gene expression [15]. #### 2.3. Transcriptome Analysis Transcriptomic data are generated in a similar way to genome sequencing libraries, except cDNA from reverse transcription is sequenced rather than genomic DNA. Aligning these reads to a transcriptome is a more complicated problem than aligning to a genome because of RNA variants, splicing, and otherwise uneven transcription of the genome. Transcriptome alignment tools require aligners such as Bowtie or BWA but require different information to annotate the transcription of the genome. The most commonly used program for transcriptome analysis is Spliced Transcripts Alignment to a Reference (STAR) [17]. This program is what is used by The Encyclopedia of DNA Elements (ENCODE), so should be used if someone wants to directly compare their results to most other experiments [18]. A newer program that is even faster than STAR is Kallisto, which is beneficial because it reduces computational expenses for very large experiments [19]. Salmon is another reputable transcriptomic software as well, among others [20]. Any of these different software algorithms will produce useful results for your experiment that may be later used for multi-omic integration. Once this software has been run, several metrics will be generated for every transcript in each sample, including transcripts per million (TPM) and reads per kilobase of transcript, per million mapped reads (RPKM). To begin your analysis, several steps need to be taken. One analysis program in particular is used because of its end-to-end capabilities: the R package DESeq [3]. Similar packages include edgeR and limma [21,22]. The normalized reads can then be used for downstream analyses listed below. To perform custom analyses, the data should be read into a data matrix, which is helped by a program such as the R program tximport [23]. In this way TPM or RPKM can be pulled out for every sample. These should then be corrected for batch effects, for which RNAseq is particularly sensitive. The program sva::COMBAT() from R is excellently suited for batch correction [24]. Once corrected, the data are ready for downstream data analysis as illustrated below. The first step in differential analysis workflow is data normalization, in order to guarantee the accurate comparisons of gene expression between and/or within samples. Proper normalization is essential not only for differential analysis, but also for exploratory analysis and visualization of data. The main factors that we often need to consider during count normalization are sequencing depth, gene length, and RNA composition. There are several common normalization methods to account for the "unwanted" variates, including counts per million (CPM), TPM, reads/fragments per kilobase of exon per million reads/fragments mapped (RPKM/FPKM), and DESeq2- s median of ratio trimmed mean of M values (TMM) [3,25]. CPM, TPM, and RPKM/FPKM are the traditional normalization methods for sequencing data, but they are not suitable for differential analysis due to the fact that they only account for sequencing depth and gene length, but not RNA composition. Accounting for RNA composition is especially crucial for the scenario with a few highly differentially expressed genes between samples—big differences in the number of genes expressed between samples, which can skew the traditional types of normalization methods. It is highly recommended to account for RNA composition, especially for differential analysis [3]. Due to this, TMM normalization was developed and can be conducted in the edgeR package [22]. DESeq2 package implements the normalization method of median of ratio [3]. The DESeq2 package implements transformations by computing a variance stabilizing transformation which is roughly similar to log2 transformation of data, but also deals with the sample variability of low counts, generating vst and rlog formats of data. However, both formats are designed for applications other than differential analysis, such as sample clustering and other machine learning applications. From these data, transcript enrichment can be performed using gene ontology (GO) or another such categorization method. GO involves assigning one or more functions to each gene based on its experimental function or categorization, and these categories are assigned to several genes. Then between the cases and controls one may see whether the GO categories are significantly enriched. DAVID bioinformatics offer a wide range of enrichment methods, including GO enrichments [26], although there are many similar GO algorithms such as GOrilla [27]. Another powerful pathway analysis and mechanism elucidation tool is ingenuity pathway analysis (IPA). It is an all-in-one, Web-based software application, enabling analysis, integration, and understanding of omics data, including gene expression, miRNA, SNP microarray, proteomics, metabolomics, etc. However, one of its downsides is that it is only commercially available. Nguyen et al. [28] systematically investigated and summarized the comprehensive pathway enrichment analysis tools, and concluded that topology-based methods outperform other methods, given the fact that topology-based methods take into account the structures of the pathways and the positions of each molecule in the biological system map. The best topology-based approaches include SPIA (signaling pathway impact analysis) and the ROntoTool R package. Deeper analyses may be performed as well. For example, from peripheral blood mononuclear cells (PBMCs) the composition of the white blood cells may be estimated from expression of marker genes using software such as immunoStates [29], although others are effective as well [30]. These data may complement integrative analyses, integrating enrichment software from various omes. #### 2.4. Metagenomic Analysis Metagenomic analysis also is similar to other nucleic acid omes. All the genetic material from a microbiome sample, often from stool, is sequenced. This review will discuss sequencing on an Illumina platform; however, other sequencing platforms are appropriate as well. This is all the genetic material from multiple organisms, hence the metagenome. These reads must be queried against a database, similarly to the previous methods. For example, the pipeline can query the human microbiome project database not just for different taxa, but also for biochemical pathways and even related individual genes [31]. This is important because taxa alone do not provide all the functional biological data about a microbial population. Such data provide a wealth of information about several levels of the microbiome. A fast, highly sensitive, although less specific method is querying chunks of the reads, or kmers, against a database, as used in Kraken. To aid with the problems presented by genomic flexibility in microorganisms, a kmer approach is increasingly being utilized, which requires only aligning part of the read, not the entire one [32]. These methods require very deep sequencing, and a more cost-effective method may be to sequence the 16s rDNA gene from bacteria [33]. This gene acts like a molecular clock, determining which taxa the read is from just like a clock determines time, with different parts of the gene highlighting different granularity in the taxonomic tree [34]. This method and metagenomic methods return count data, where read depth is used as a measurement of how abundant that particular part of the microbiome is. Methods to determine absolute abundance, not just relative abundance, should be used as well—for example, spiking a known amount of a microbe or DNA into the sample. Once the count data has been determined, it may as well be batch corrected. From these data, microbes with known importance or microbial pathways with known biological relevance to the host may be determined using methods described below. #### 2.5. Mass Spectrometry for Biomolecules Like for the nucleic acid methods, mass spectrometry (MS)-based methods share some similarity, but also have their unique properties. Each ome is first fractionated through liquid chromatography (LC), the parameters of which are determined according to its own unique biochemistry properties. In proteomics, the proteins are typically digested into shorter peptides first. After LC, proteins are sent through data dependent MS/MS or data independent acquisition. The software for calling peaks depends on the platform used, with the most popular being Skyline and Perseus [35,36]. Data-dependent acquisition generates more identified proteins but is less comparable between samples. Each method requires its own data analysis software to call peaks, for example, openSWATH for the data independent acquisition [37]. Furthermore, even with data from a single piece of software, the library compared against is absolutely essential for data quality. For example, the TWIN library may produce particularly good data with the openSWATH platform [38]. Metabolomics data can be generated in different platforms, such as reversed RPLC-MS (reversed phase liquid chromatography-mass spectrometry), HILIC-MS (hydrophilic interaction chromatography-mass spectrometry), and so on. These are then imported into Progenesis QI 2.3 software which is able to convert spectra data to data matrix for further downstream analysis. Further data preprocessing steps include but not limited to filtering noise signals, data imputation, retention time adjustment, and data normalization [39]. Removing batch effects is one of the most crucial tasks in metabolomics, and various classic and advanced methods have been developed. Recently, Feihn et al. published a random forest model-based normalization method SERRF (systematic error removal using random forest), and the authors claimed that this method outperforms other normalization methods, including median, PQN, linear method, and LOESS [40]. This normalization can be applied on both untargeted metabolomic and lipidomic datasets. After data cleaning, one can use either an in-house metabolite library or public databases (HMDB, Metlin, MassBank, NIST, etc.) for metabolite annotation. With different available data, the annotation needs to be defined clearly with confidence levels. Our laboratory uses a Lipidyzer, a semi-targeted lipidomics platform, to determine the lipid absolute abundance by using lipid chemical standards [41]. The software that calls the lipid species is LWM (Lipidomics Workflow Manager), although this area is a fertile one for growth. From these methods, individual molecules, proteins, and lipid species may be associated with biological questions of interest. Furthermore, in the lipidomic data classes of molecules may be enriched from their individual species. For example, triacylglycerols as a whole, not just individual triglyceride species, may be associated with the biological question of interest. Proteomic and metabolomic data enrichments may be performed with DAVID or MetaboAnalyst [42]. Ingenuity pathway analysis (IPA) from Qiagen may also be used for enrichments of the proteomic and metabolomic data, and it may also be used to integrate the two together. The Kolmogorov–Smirnov method is an alternative approach for pathway/chemical class enrichment analysis in the metabolomics and lipidomics field, which is able to use ranked significance levels as input. There are many other pre-written computer programs available, such as IPA, to analyze multi-omics data (reviewed in [43]), but we will focus on methods for developing your own customized pipeline, rather than pre-built Web-based software. From all these individual methods, information is gleaned about that particular omic measurement. Furthermore, these methods all generate data structured similarly that facilitate omic integration. They all generate a list of analytes for every sample, be it a transcriptome, microbiome, proteome, lipidome, or metabolome. These analytes are then associated with a particular intensity (Figure 1). There are many differences between these omic measurements, but this similarity in data structure facilitates downstream analysis (Figure 1). There are many other omics measurements that share similarities with those mentioned, and there are numerous databases containing already-generated datasets, which may also be used for integrative multiomics rather than generating new data [44]. For example, http://educationknowengorg/sequenceng/, mentions 68 different next-generation sequencing technologies, most of which are omics measurements. Nonetheless, most share similarities with those already discussed here. Figure 1. The molecules profiled in multi-omics studies. We describe 6 levels of information, starting from the bottom to the top: genome, epigenome, transcriptome, proteome, metabolome, and metagenome. The genome, epigenome, transcriptome, and metagenome are profiled by sequencing-based technologies such as sequencing by synthesis, depicted here, to profile a comprehensive set of nucleic acid molecules. On the other hand, mass spectrometers generate proteome and metabolome profiles as depicted here through measurements of biomolecules' masses and charges. For overlapping technologies, each omic level provides unique information and insights into cellular activity present in conditions being studied. By leveraging the layers of information, longitudinal and cross-sectional multi-omics studies find modules (e.g., cell signaling pathways) that are differential between healthy and disease states. These modules represent complex system biology networks that give precise insights into the molecular dysregulation in disease states. #### 3. Designing a Quality Study The first step in understanding an analysis flow for integrative multi-omics is determined by your study design. Cross-sectional and association studies are beneficial in their relative ease to implement, and their ability to generate large amounts of data (Figure 2a). Typically, cross-sectional studies do not involve a randomized intervention, precluding causal inference. They involve taking a population split between cases and controls, and then sampling them evenly, and are excellent methods for determining associations. Conversely, longitudinal studies are relatively difficult to recruit large numbers of participants to because they generate large numbers of time points and become expensive. However, the longitudinal nature increases the statistical power of a relatively small number of participants [45] (Figure 2b). Longitudinal studies further facilitate making causal inferences and allow for more accurate predictions. Each study design, with its strengths and weaknesses, has a slightly different flow of analysis. Wherever possible, the multiple omic measurements should be selected not staggered in time. For example, if the treatment course is seven days, all the participants should be sampled on the same days during treatment. This will greatly facilitate the analysis methods. Some advantages of longitudinal studies include the ability to associate events chronologically with particular interventions or exposures. They allow a study of change over time, or a delta measurement from baseline, as discussed below, which can be more powerful than studying a single point in time for the effects of an exposure or intervention. They also allow for establishing the chronological order of events, which is essential for establishing causation—again, something that is precluded in cross-sectional association studies. There are relatively few negative effects other than the difficulty of recruiting large numbers of participants, but they may also include loss of individuals over time, confounding results [46]. Figure 2. Typical multiomic study designs. Gray dots represent samples taken. (a) A case control observational study. A population is taken with participants that have the phenotype of interest (cases) and those without (controls). Cases and controls are sampled in even amounts. (b) A randomized longitudinal study where n participants are randomized into two arms of a study. In this case an increasing treatment dose is administered, and samples are taken every week. In each individual, there are apparent biases in the technologies and analytical methods, which limit insights into biology. Often signals from individual omes are difficult to label as accurate or relevant because the information does not connect to the broader context of the system. Multi-omic integration offers an opportunity to use orthogonal methods to measure the same molecular pathways and processes. Such methods partially mitigate the inherent false positives and false negative rates in the single omes, as finding the similarities and biological connections supports the truly biologically-relevant information [47]. #### 4. Analysis Methods for Multi-Omic Integration #### 4.1. Dimensionality Reduction The first step in an omics study is to reduce the dimensionality of your data so they can be visualized. In a metagenome, for example, there may be hundreds of microbial species. This means that every sample is a data point with hundreds of dimensions. Dimensionality reduction techniques will take the data and reduce them to fewer dimensions, often as few as two or three, that represent most of the variation in the data. Then it is easier to visualize and use statistics that require fewer dimensions. The first dimensionality reduction technique invented is principle component analysis, which is a widely used unsupervised method. This method, though yielding valuable results, is not the most statistically precise because it assumes normally distributed data. Anyone who works in omics will testify that the data are never normally distributed, although transformations can make the data approximately normal. One superior method is non-metric multidimensional scaling. This method is iterative and nonparametric, avoiding problems with unusual distributions, and it handles zero-truncated data well—a phenomenon in which in some samples a particular analyte is undetectable and in others it exists at a high level. Another method, tSNE, is particularly well designed to separate well defined groups. Besides t-SNE, UMAP (uniform manifold approximation and projection) [48] is a newly developed dimension reduction technique for non-linear relations. It usually implements faster than t-SNE, especially when it concerns large number of data points or a number of embedding dimensions greater than 2 or 3. It has many applications in single-cell sequencing data. Other methods include principal coordinate analysis and multidimensional scaling. Every method is capable of providing useful information; however, properly selecting a method can increase your statistical power. The information gleaned from dimensionality reduction is similar across omic techniques. It can discover batch effects, particularly in mass spectral data. If two batches do not overlap, then additional correction techniques need to be applied. This method can find samples that failed, which would be represented as outliers in the data. Once data quality has been established, these methods can find any structure in the data that might be associated with biologically relevant variants. This is the most basic example where a metadatum, participant ID, may be grouped together. However, there are many more—sex, insulin resistant status, etc. In the case of the microbiome, it can also be used to measure beta diversity, as outlying samples will have different microbial compositions than the rest of the cohort. #### 4.2. Normalizing the Data Once the structure of the data has been determined, omics measurements can be grouped together for integration. Usually they are done so after log, log2, or other transformations to facilitate downstream statistics [49,50]. The log transformation is normally used to make highly skewed data approximately approach a normal distribution. This can be useful both for facilitating the data to meet the assumption of statistic models and for making patterns in the data more interpretable. Microbiome data are so unusually distributed, other transformations may be applied, such as arcsin. With certain longitudinal designs they can be normalized to the baseline measurements to only measure the deltas from the baseline, reducing the effects of inter-individual variability. This is absolutely essential in longitudinal data to reduce the effects multiple individuals would have on biasing a sample, and is one of several strengths of that study design. A z-score is another normalization method that standardizes all the analytes to the same range. This alleviates the problem of vastly different expression levels, facilitating grouping several different omes together for integration. For example, if one wanted to integrate the metabolome and gut microbiome, the values for the metabolome may be in the tens of millions, while analytes in the microbiome may be zero truncated, with most values being 0. To compare these two, particularly visually, they must be on a similar scale. Z-scoring makes the average value for every analyte 0, and then one standard deviation above that 1, etc. #### 4.3. Correlation Networks Analyses Once these normalizations and transformations are performed, correlation metrics can inform one about the most basic relationships between the analytes. Pearson correlation coefficients (PCC) and spearman correlation coefficients (SCC) are the two most typical types of correlation metrics. The PCC is a parametric metric with more accuracy, whereas the SCC is more robust if outlier samples are present. One should target analytes of the most interest (e.g., only the significant molecules) if possible, because with too many analytes in networks, it is difficult to capture the most useful biological information and it is inclined to be masked by the underlying noise. Correlation networks are much more effective when dealing with deltas in longitudinal data that reduce interindividual variability. If more than one sample, not corrected to baseline, is from a single individual, such an analysis will be overfit and produce false positives. Additionally, one must always correct for multiple hypotheses during these projects to reduce false positives. In these data a Benjamini–Hochberg correction is appropriate. One may also use a Bonferroni correction, but in some omic studies that may overcorrect, losing true positives. Both will have their uses and may be differentially used in longitudinal baseline normalized vs. unnormalized data. This correlation analysis can be plotted as a network diagram, which is a fantastic visualization tool for this type of data. Though high-level visualizations, network diagrams offer compelling, informative overviews of interactions in biological systems [51]. When comparing interaction networks across different conditions, disease states, or interactions, a network analysis may provide you appropriate information about how the two states differ. A network analysis will provide one with total nodes (analytes) that are connected in the network, the total number total edges between the networks (significant correlations), and many other important relationships, such as the numbers of positive and negative correlations. Complementing visualizations, these summary statistics provide an excellent overall view of the co-correlations occurring in any multi-omic project. This type of topological analysis is not only able to provide practitioners straightforward and clear ideas when comparing multiple networks, but also provide insights into network hubs and centers, which may have many applications in drug target selection and identification of key regulators. There are several packages for R—igraph, statnet, ggnetwork, ggnet, ggraph, etc.—with highly related functionalities that perform these analyses [52,53]. R packages "igraph" and "statnet" are able to provide quick visualizations, which are good for a quick exploration about the network structure but are not necessarily the most efficient ways for aesthetically perfect visualization. R packages "ggnet" and "ggnetwork" are very similar packages, and both seem to use a variant of the ggplot syntax, meaning that they would be advantageous if you are familiar with the ggplot system. #### 4.4. Cross-Sectional Analyses and Testing Categorical Variables In a cross-sectional study, when testing a single analyte between two sets of samples, the nonparametric version of the student's t-test, Wilcoxon rank sum test, is appropriate. A t-test assumes a normal-like distribution and should be used with care, as omics measurements are extremely rarely Gaussian. If confident that prior information will be obtained before the test, Bayesian counterparts to these tests will provide more power. These are not necessary, and should only be used by an expert. In a cross-sectional study where two categories are being tested against, one may further use logistic regression as a means of regressing between these categories. This regression fits a curve to binary data, generating an odds ratio and p-value. When analyzing across more than two categories of data, one should use the non-parametric analysis of variance (ANOVA), Kruskal–Wallis. This method may be used to test a trend in your data over categorical variables. When correcting for multiple variables, one may use a multivariate ANOVA (MANOVA), but this should be used with care because ANOVAs assume a normal distribution. To avoid these assumptions about distributions, a permutational multivariate analysis of variance (PERMANOVA) should be used. #### 4.5. Testing along Continuous Variables Another method determining trends over categorical variables is multiple linear regression. Like ANOVA, this may be used to find trends in one or more categorical variables. However, multiple linear regression can find trends over continuous variables as well, or any combination thereof. Although multiple linear regression also assumes a normal distribution, it can still be a valuable tool for detecting trends in data and is widely used by multi-omic researchers. In cases like this, where the statistical assumptions do not perfectly match data distributions, orthologous methods should be used for confident assessments. Even more sophisticated than multiple linear regression is a mixed model. These can find trends in data and can also find the variance in data for random variables. Random variables are those that are randomly distributed in your data, say a random assignment of sex, so they are not associated with the outcome variable. Nonetheless, these variables can add variance, making the data noisier. Further, these mixed models can select other distributions than Gaussian, such as Poisson, so variables that violate normality may be modeled better. Mixed models are appropriate to account for complicated and heterogeneous datasets with confounders—gender, race, age, BMI, etc. Mixed models are particularly well suited for tracking longitudinal data [45]. Together, these methods are powerful for detecting trends in the data. #### 4.6. Clustering Algorithms Clustering algorithms group similar samples or analytes together. Two primary clustering algorithms are hierarchical and k-means clustering. These are "hard" clustering algorithms which force analytes or samples into particular groups. This may be useful to determine whether samples cluster by individual, batch, or some other biological measurement, for dimensionality reduction techniques. They can also be used to determine outliers in the data, which may be of special interest to the researcher. To find trends in longitudinal data, fuzzy c-means clustering is a powerful tool. The R mfuzz package provides tools for this analysis [54]. This is a "soft" clustering algorithm, giving analytes a score known as membership in every cluster, rather than forcing them into a single cluster. However, like other clustering algorithms, it still finds analytes with similar expression profiles. Using the previously mentioned z-scores, c-means clustering finds longitudinal trends in data for multiple omic measurements. These trends are powerful if one wants to find dose, temporal, delayed, or other response patterns in multi-omics data [55,56]. One of the most critical and haunting issues in clustering is to determine the optimal number of clusters. Selecting an inappropriately small number of clusters would cause the missing detection of some meaningful molecular trends and clusters, whereas an improperly large number of clusters may result in redundancy of cluster detection. There are several ways to assist the selection of the optimal number of clusters. One of the classic methods is called the elbow method, which calculates the within-cluster sum of squared error (wss). This method is widely applied; however, it gets tricky to determine the "elbow" point. Another way to survey this issue is to calculate minimal centroid distance, which is similar to the elbow method, aiming to find the "elbow" point to gain the minimal centroid distance. Another more efficient method is to calculate the correlations between cluster centroids, and decide on the optimal number of clusters once high positive correlations (e.g., 0.85) are detected. Another method of clustering, supervised clustering, involves placing a priori information into a model before using the clustering algorithm. For example, if you have cases and controls, these may be entered into the data beforehand, or if you have longitudinal data with doses, the baseline controls may be contrasted with the doses. Categorical variables are required for this type of clustering, but they are an excellent method of assuring one will find analytes with similar expressions in the data [46]. #### 4.7. Feature Selection for Covarying Analytes A powerful tool in the arsenal of multi-omics researchers is feature selection. In some data, the analytes strongly covary. For example, in the metagenome, if one organism increases it will have an effect on every other organism in the system. In such circumstances it may be difficult to know which of these analytes to prioritize putting in a model. Least absolute shrinkage and selection operator (LASSO) and ridge regression tackle these problems. These functions will weight or eliminate the variables with the most and least explanatory power in your model. This way, future analyses may be performed on more manageable and more meaningful data, which may also increase statistical power. There are numerous feature selection methods, and descriptions and comparisons of all of them are obviously beyond the scope of our review. We mainly highlight two of them (LASSO and ridge) because they are widely applied penalized algorithms that reduce model complexity and prevent over-fitting which may result from simple linear regression. The main principle of these two regularization methods is to restrict or shrink the coefficients towards zero for the non-impactful features, in order to reach the goal of feature selection. #### 4.8. Machine Learning Machine learning is an important subset of artificial intelligence, and nowadays has drawn attention in various fields. In omics studies, machine learning is widely applied on classification and prediction problems by using omics profiling data. Different suites of machine learning algorithms are suitable for classification and prediction scientific problems. Classification and prediction, as two main branches of machine learning, depend on the types of tasks or problems that are intended to be solved by machine learning and are either categorical (classification) or continuous (prediction). There are three main types of machine learning algorithms: unsupervised, supervised, and reinforcement learning. Classification and regression are the two main prediction domains in the machine learning field. Classification is the problem of predicting a discrete class output, while regression is to predict a continuous quantity output. Due to the pronounced differences in principles for these two domains, the modeling algorithms applied on these two problems are different. Some algorithms can be used for both with minor modifications, e.g., decision trees and artificial neural networks, whereas some algorithms are only suitable for either classification or regression problems—e.g., logistic regression can only be used for classification, and linear regression is only for regression predictive modeling. More importantly, the matrices that are used to evaluate models varies for classification, e.g., accuracy, are usually used for assessing classification models but not regression algorithms, whereas root mean squared error (RMSE) is only for regression predictive models but not classification models. One of the useful supervised machine learning algorithms in multi-omics is the random forest. A random forest is not a black box telling you which parameters are the most predictive of biology. Conversely, neural networks and deep learning are typically not appropriate for multi-omics datasets because of the structure of the multi-omics data, normally with more variables than sample size. Neural networks provide more accurate predictions when there are many samples, and relatively few measurements per sample. Multi-omic studies are typically the opposite, with relatively few samples but many, many measurements per sample. There is nothing in principle preventing neural networks from working on multi-omic datasets, but rather the practical considerations of how these studies are designed. Further, neural networks and deep learning are "black boxes" where the decisions of the algorithm are unknown to the researcher. For these reasons, random forests may provide better predictions in multiomis data, as measured by recall, area under receiver operating receiver curve, and the Mathews correlation coefficient [57]. Though more sophisticated than other analysis methods, these machine learning techniques are phenomenal for first, exploratory, unbiased passes on the data. They will determine which features are most predictive of data outcome, and what to look for as grounding during other analyses. #### 5. Conclusions There are several limitations in multi-omic integration, including potential statistical overfitting, varying distributions between analytes, and limitations in throughput for some techniques [43,58,59]. Nonetheless, multi-omics are a suite of tools that allow researchers to answer questions with unparalleled depth. These measurements are not perfect in themselves, and consistency between omic measurements will ensure the discoveries are true to the underlying biological reality. Furthermore, there are no perfect methods for analyzing these data. A researcher should be confident in their findings when their discovery comes up in multiple omes but also when discovered through multiple analysis and statistical methods. What we have discussed here is not exhaustive of the excellent analysis methods that exist, but we are confident that any researcher employing these techniques will find the trends present in their multi-omic dataset successfully. Author Contributions: Conceptualization, S.M.L., A.S., S.W., and M.P.S.; writing—original draft preparation, S.M.L.; writing—review and editing, S.M.L., A.S., S.W., and M.P.S.; visualization, S.M.L., and A.S.; supervision, M.P.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We would like to acknowledge Brittany Ann Lee and Jeinffer V. Quijada for the consultations they provided during writing. Conflicts of Interest: The authors declare no conflict of interest. #### References Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). ## Article From Prevention to Disease Perturbations: A Multi-Omic Assessment of Exercise and Myocardial Infarctions Melanie T. Odenkirk 1, Kelly G. Stratton 2, Lisa M. Bramer 2, Bobbie-Jo M. Webb-Robertson 3,4, Kent J. Bloodsworth 3, Matthew E. Monroe 3, Kristin E. Burnum-Johnson <sup>5</sup> and Erin S. Baker 1,6,\* Abstract: While a molecular assessment of the perturbations and injury arising from diseases is essential in their diagnosis and treatment, understanding changes due to preventative strategies is also imperative. Currently, complex diseases such as cardiovascular disease (CVD), the leading cause of death worldwide, suffer from a limited understanding of how the molecular mechanisms taking place following preventive measures (e.g., exercise) differ from changes occurring due to the injuries caused from the disease (e.g., myocardial infarction (MI)). Therefore, this manuscript assesses lipidomic changes before and one hour after exercise treadmill testing (ETT) and before and one hour after a planned myocardial infarction (PMI) in two separate patient cohorts. Strikingly, unique lipidomic perturbations were observed for these events, as could be expected from their vastly different stresses on the body. The lipidomic results were then combined with previously published metabolomic characterizations of the same patients. This integration provides complementary insights into the exercise and PMI events, thereby giving a more holistic understanding of the molecular changes associated with each. Keywords: lipidomics; metabolomics; multi-omics; planned myocardial infarction (PMI); myocardial infarction (MI); exercise; heart; cheminformatics #### 1. Introduction For decades, physical activity and diet have been considered the primary preventative strategies for numerous diseases, including cardiovascular disease (CVD). As the leading cause of death worldwide, rigorous characterization of CVD and the subsequent incidences of myocardial infarction (MI) are crucial for reducing its occurrence [1]. Despite the prevalence of CVD and resulting MI events worldwide, the complex pathophysiology underlying CVD origins has yet to be fully defined [2]. Even with advancements such as diagnosis with CK-MB and cTn assays and methods for CVD prediction from traditional risk factors alone or in tandem with molecular predictors, CVD-related events continue to be the leading cause of death worldwide [1,3–5]. Thus, improving our understanding of these disease mechanisms could serve to reduce the current morbidity rate of CVD by providing more effective prevention, intervention and treatment strategies. In CVD and other diseases, such as type 2 diabetes, osteoporosis and some forms of cancer, there is a well-recognized, negative correlation with the intensity, duration and continuation of exercise events [6–8]. Since exercise subjects the heart to hemodynamic Citation: Odenkirk, M.T.; Stratton, K.G.; Bramer, L.M.; Webb-Robertson, B.M.; Bloodsworth, K.J.; Monroe, M.E.; Burnum-Johnson, K.E.; Baker, E.S. From Prevention to Disease Perturbations: A Multi-Omic Assessment of Exercise and Myocardial Infarctions. Biomolecules 2021, 11, 40. https://doi.org/ 10.3390/biom11010040 Received: 24 November 2020 Accepted: 24 December 2020 Published: 30 December 2020 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). stress and overloading of pressure and volume [8], morphological adaptation of the heart occurs following recurrent exposure to exercise, effectively diminishing the risk of heart disease by reducing cholesterol and suppressing hypertension and atherogenesis [9,10]. However, over-exertion of the heart muscle from exercise can result in calcification that limits the capacity of the heart to pump blood, thereby increasing the risk of cardiovascular events [11]. On the other hand, a sedentary lifestyle along with high blood pressure, abnormal blood lipid profiles, smoking and obesity are all major risk factors for CVD, typically triggering the development of an intermediate phenotype prior to a MI [12–14]. Therefore, elucidating a balance between the beneficial and detrimental mechanisms of exercise is crucial for optimizing heart performance and reducing CVD risk and mortality rates [13]. While exercise and diet can be preventative, certain people are genetically predisposed to CVD and other heart diseases. Therefore, leveraging models of stroke and MI events responsible for 80% of CVD end-stage phenotypes provides additional molecular information about treatments and the induced injuries. Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiac disease, estimated to be present in 1 out of every 500 individuals [15,16]. Obstructive HCM (HOCM) is a subtype mechanistically defined by the barricaded outflow of the left ventricular heart cavity at rest (1/3 of cases) or at provocation (1/3 of cases) [15,16]. The reduction of left ventricular outflow in HOCM cases culminates in increased left ventricular pressure, high wall stress, impaired left ventricular filling, myocardial ischemia and a reduced cardiac output [16,17]. Currently, aspirin, β-blockers and pacemakers are all common remediation strategies to mitigate these symptoms [16,17]. Failure of these therapeutic approaches to alleviate left ventricular blockage, however, requires removal of obstructing tissues through either surgical excision or alcohol septal ablation (ASA), where an injection of alcohol triggers a planned myocardial infarction (PMI) and reduces the left ventricle blockage caused by systolic anterior motion of the mitral valve [16]. While both procedures have had similar patient outcomes and survival rates, ASA treatment and the resulting PMI have proven to be a less invasive approach preferable for surgically at-risk patients [16,17]. Evaluating the molecular changes occurring from a PMI also grants researchers tremendous insight into the pathophysiology of spontaneous MI events that plague one American every 40 s with a global mortality rate of CVD-related events accounting for 31% of the deaths in 2015 [18,19]. Mass spectrometry (MS) has become a popular analytical tool to characterize molecules changing in biological systems through omic studies. While the annotation of a singular "ome" (i.e., proteome, lipidome, metabolome) elucidates significant aspects of disease pathophysiology, comprehensively characterizing a disease through one class of biomolecules does not provide the holistic information often needed. Thus, multi-omic measurements, wherein multiple classes of biomolecules are analyzed and integrated, provide a greater understanding of molecular interplay and pathophysiology [20]. For example, since metabolites and lipids both reflect immediate changes occurring in a system, together they allow for an investigation into early-stage perturbations [21,22]. Furthermore, lipids have routinely been linked to exercise and MI mechanisms [2,23–25], so their combination with metabolites provides a complementary way to assess system dysregulation. In this study, lipidomic assessments were performed on plasma taken from two cohorts; the first cohort's samples were taken before and one hour after exercise performed with a specific treadmill testing procedure, and the second cohort's samples were taken before and one hour after a PMI. The lipidomic results were then compared to a targeted polar metabolite study of the same patient cohorts [26,27], and together, the multi-omic comparison provided a more comprehensive characterization of the various biomolecule classes altered upon different stressors of the body and heart. This comparison therefore allowed for the exploration of molecular differences between CVD-related events and preventative strategies within humans. #### 2. Materials and Methods 2.1. Sample Extraction and Data Collection 2.1.1. Human Sample Collection and Extraction Both an exercise and a PMI cohort were evaluated in this manuscript, and informed consent was obtained from all human participants in the studies. In the exercise cohort, plasma samples were collected from the periphery veins of 25 patients before and one hour following exercise treadmill testing (ETT) [26]. In the PMI cohort, plasma samples were also collected from the periphery veins of an additional 20 patients before and one hour following a PMI [27]. The paired before and after sampling of the same patient for both studies was performed to yield a high statistical power despite the limited number of samples analyzed, since the before sample could be used as the control for each patient [28]. An overview of the patient demographics for both the ETT and PMI cohorts is given in Figure 1 and Supplemental Table S1. Additional cohort information is also expanded upon in the original publications [26,27]. Notably, the male demographic of the exercise cohort was large compared to the PMI study, wherein women were in the majority [26]. Additionally, in the exercise study, enrolled patients had to meet a normal exercise tolerance criteria, which included having an estimated peak VO2 capacity over 70%, a heart response rate exceeding 85% predicted baseline and a pre-exercise fasting period of4h[26]. PMI patients were also monitored with CK-MB and troponin T assays, with peak levels observed at standard spontaneous MI times with CK-MB at 4.5 h and troponin T at 8 h following a PMI event [27,29]. The PMI derivation cohort were all primary HOCM cases with septal thickness ≥16 mm; resting outflow tract gradient ≥30 mmHg; inducible outflow tract gradient ≥50 mmHg; failed medical intervention; and appropriate coronary anatomy [27]. Targeted analysis of 210 metabolites was completed in the original publications for each cohort with a triple quadrupole mass spectrometer (AB4000Q; Applied Biosystem/Sciex, Farmingham, MA, USA), and detailed protocols on those methods can be found for the ETT study in Lewis 2010 [26] and PMI study in Lewis 2008 [27]. Figure 1. Demographics for the PMI cohort (left) and ETT cohort (right). Continuous variables are given as mean ± standard deviation and categorical variables are shown as percentages. #### 2.1.2. Lipid Extraction For the lipidomic study, lipids were extracted in 2 mL Sorenson tubes from 25 μL aliquots of plasma following a modified Folch protocol [30,31]. Briefly, 600 μL of a 2:1 mixture of −20 ◦C chloroform/methanol was introduced to the plasma samples which was then vortexed for 30 s. A phase separation was induced by adding 150 μL aliquots of HPLC grade water and then vortexed again for an additional 30 s. The samples then rested for 5 min at room temperature prior to centrifugation at 12,000 rpm for 10 min at 4 ◦C. Samples were then placed on ice where 350 μL aliquots of the bottom organic layer were removed, dried in a speedvac and then re-suspended in 250 μL of 2:1 chloroform/methanol for storage at −20 ◦C. Immediately before instrumental analysis, the total lipid extracts were dried down and reconstituted in 5 μL chloroform and 100 μL methanol. Pooled case and control samples for the exercise and PMI studies were generated by combining 5 μL aliquots of each before plasma sample separately. #### 2.1.3. Lipidomic Instrumental Analysis Lipidomic instrumental analysis of the 45 before and 45 after extracted human plasma samples was completed with an Agilent 6560 IM-QTOF MS platform (Santa Clara, CA, USA) outfitted with the commercial gas kit (Alternate Gas Kit, Agilent, Santa Clara, CA, USA) and a precision flow controller (640B, MKS Instruments, Andover, MA, USA). The LC– IMS–CID–MS data were collected in both positive and negative ESI from 50–1700 m/z with a 1 sec/spectra cycle time. Reverse phase liquid chromatography (RPLC) separation was completed with a 10 μL sample injection onto a Waters CSH column (3.0 mm × 150 mm × 1.7 μm particle size) on a Waters Acquity UPLC H class system (Waters Corporation, Milford, MA, USA). Separation of lipid species was achieved with a 34-min LC gradient (mobile phase A: acetonitrile/water (40:60) containing 10 mM ammonium acetate; mobile phase B: acetonitrile/isopropyl alcohol (10:90) containing 10 mM ammonium acetate) at a flow rate of 250 μL/min as described in Table 1. A 4-min column wash and 4-min equilibration were also used as described in Table 2. Table 2. Lipid column wash. #### 2.2. Data Processing #### 2.2.1. Lipid Identification Accurate mass tag (AMT) matching within LIQUID software was used to assign all lipid identifications [32]. The LC–IMS–CID–MS platform typically allows for the assignment of head group and fatty acyl (FA) structural moieties of each uniquely identified lipid species using the criterion of mass accuracy below 5 ppm, precursor and fragment peak alignment across dimensions, and CCS values < 2% different from the reference value. While head group annotation is largely unambiguous, FA assignment is more complex due to the propensity of isomers. From the collision induced dissociation (CID) measurements, the number of carbons and double bonds is generally achieved; however, additional specifics, such as sn-backbone position, double bond position or double bond orientation, are often indistinguishable in these studies [33]. Therefore, the most confident lipid speciation achieved through this analysis included the head group and individual fatty acyl groups with unknown sn-positions, as denoted by "\_" (i.e., PC (16:0\_18:2)) [34]. For lipids where individual FA constituents could not be identified, the summed carbon and double bond counts are noted, e.g., PC (34:2). Any features matching more than one lipid identification are separated by a ";" to denote both as potential matches. Furthermore, isomeric experimental observations were assigned "\_A"; "\_B"; etc., to denote the observed chromatographic and/or IMS separation of these species. The peak areas of the 352 lipids identified in the exercise study (262 from positive mode, 85 in negative mode and 5 in both modes) and the 299 lipids identified in the PMI study (225 in positive, 72 in negative and 2 in both modes) were exported as a ".csv" format for processing and statistical assessment regarding each before/after paired comparison (Supplemental Tables S2 and S3). #### 2.2.2. Data Processing and Statistics Statistical analysis of the targeted polar metabolites was carried out as detailed previously [26,27]. Briefly, in the targeted annotation of 210 metabolites in the ETT and PMI studies, 20 were found to be statistically significant one hour following exercise (16 upregulated and 4 downregulated) and 13 were statistically significant one hour following a PMI (7 upregulated and 6 downregulated) at a Benjamini–Hochberg corrected p ≤ 0.005 cut-off. Processing and statistics of the lipidomics data also followed the same procedures, where statistical significance was determined from log2 transformed abundances using MetaboAnalyst (version 4.0, Edmonton, AB, CA) [35]. The ETT statistical analysis was completed using a paired t-test, and the PMI comparisons were completed with a Wilcoxon signed-rank paired t-test due to their unequal variance. A Benjamini–Hochberg multiple comparison correction was also applied for both analyses with a significance cut-off of p ≤ 0.005 to match the previously published metabolite statistics [36]. Interestingly, no statistically significant lipids were observed one hour following ETT, whereas the PMI study yielded 207 statistically significant lipids: 66 upregulated and 141 downregulated (Supplemental Tables S2 and S3). Comparison of sex in the PMI cohort and ischemia in the ETT cohort was completed to account for additional differentiation following the above protocols for each cohort. No significant species were detected from either comparison. #### 2.3. Data Interpretation #### 2.3.1. Lipidomics Data Interpretation Lipidomic relationships were investigated using cheminformatics to interrogate structure-function associations across head groups and fatty acyl (FA) moieties [37–39]. Head group clustering was completed with the SCOPE toolbox [39]. Here, SMILES [40] obtained from LipidMaps [34] for each lipid identification were clustered by structural similarity using an ECFP\_6 fingerprint [41], Tanimoto distance and complete linkage using the fingerprint and ggtree packages in R (Version 3.6.2, Vienna, Austria) [42,43]. Lipids with multiple LipidMaps matches were cataloged by a representative SMILES for hierarchical clustering. To facilitate the visualization of head-group trends, pigmentation of dendrogram nodes was used to denote lipid classes. FA tail presence was further assessed by selectively parsing out lipids by FA composition. For our analyses, most sn-1 and sn-2 fatty acyl positions were unknown, so all possible positions were considered to account for potential sn-positional effects. Lipids with multiple identities were partitioned into all possible identifications to visualize each potential FA contribution to significance. Summary statistics (adjusted p-value, log2 fold change) of lipids were subsequently overlaid with the pheatmap package in R [42,44]. Color gradients of red (upregulated) and blue (downregulated) were applied to visualize significance with darker colors indicating a larger fold change (log2FC) or smaller p-value (adjusted p-value), while grey values represented identified but not statistically significant lipids. #### 2.3.2. Multi-omics Data Interpretation Hierarchical clustering was again utilized to assess the multi-omic association of statistically significant metabolites and lipids. Dendrograms provided visualization of the structurally similar and statistically significant species (BH adjusted p-value ≤ 0.005), both individually and in tandem. Metabolite clustering was accomplished with MAACS keys fingerprint, Tanimoto distance and complete linkage using fingerprint and ggtree packages in R (Version 3.6.2, Vienna, Austria) from each SMILES representation [42,43]. The resulting metabolite dendrograms allowed for a summary of the significant species following exercise and PMI events where adjusted p-values followed the same gradient as described above. Node color in the metabolite dendrogram was used to annotate the biological roles attributed to each metabolite. Conversely, in the multi-omics dendrogram built using ECFP\_6 fingerprint, Tanimoto distance and complete linkage, all metabolites were grouped together in a single node color because of the relatively small number of statistically significant metabolites relative to lipids. #### 3. Results The previous targeted metabolomic study for both the ETT and PMI cohorts provided great insight into statistically significant polar metabolites [26,27,45], but overlooked important nonpolar molecules changing due to each event. The recent annotation of lipids in both CVD and exercise has elucidated the critical roles these molecules serve in each event [24,46–56]. Therefore, the inclusion of lipidomic and multi-omic assessments in this manuscript provides a more in-depth profile of ETT and PMI molecular mechanisms. #### 3.1. Lipid Identifications and Statistical Significance To perform both the ETT and PMI lipidomic analyses, multi-dimensional assessments were carried out by leveraging a LC–IMS–CID–MS instrumental platform [32,38]. The LC– IMS–CID–MS analyses yielded a total of 352 unique lipid identifications for the ETT cohort and 299 for the PMI cohort across the same five lipid categories: glycerolipids, sphingolipids, phospholipids, fatty acids and sterols [57]. The 352 ETT lipids were composed of 216 phospholipids, 88 glycerolipids, 39 sphingolipids, 5 sterols and 4 fatty acids (Figure 2a, left); the PMI cohort had 185 phospholipids, 71 glycerolipids, 31 sphingolipids, 7 sterols and 5 fatty acids (Figure 2a, right). The breakdown of lipid category designation into classes showed both studies having: three phospholipids (phosphatidylinositols (PIs), phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)), three sphingolipids (sphingomyelins (SM), ceramides (Cer) and hexose ceramides (HexCer)), two glycerolipids (triacylglycerolipids (TGs) and diacylglycerolipids (DGs)), one sterol (cholesteryl ester (CE)) and one FA (carnitine) (Figure 2b). Additional diversity within the phospholipids was observed in the FA linkages (including alkenyl ether (plasmalogen; P-) and alkyl ether (O-)) and FA numbers (e.g., lyso and diacyl species). Only a few lipid species were specific to each cohort including a ganglioside (GM3) belonging to the sphingolipid category in the ETT cohort and a monoacylglycerol (MG) from the glycerolipid category observed in the PMI cohort. Figure 2. Identified lipid category and class coverage. (a) Five lipid categories were observed for plasma from patients in both the ETT (left) and PMI (right) cohorts. (b) In the class breakdown, the majority of the lipids fall within the sphingolipid, glycerolipid and phospholipid categories. Of the identified lipids, a drastic difference was observed in statistical significance for the exercise and PMI cohorts. One hour after ETT, no lipids were found to be statistically significant, even after further assessment of metadata, including gender, age and BMI. However, we do note our statistical criteria were very stringent to compare them with the previous metabolomics studies, so directly above our significance cutoff we observed lipids of interest within the lyso PC, GM3, PE P-, DG and carnitine classes. Specifically, we noted the largest fold changes for PC (20:5\_0:0), carnitine (10:1) and carnitine (14:1), which had values of −1.28, −1.29 and −1.18 FC. The lipidome changes in PMI, however, told a completely different story. An hour after a PMI, 207 lipids (69% identified) were statistically significant, even with the stringent criteria, with 141 downregulated and 66 upregulated (Figure 3a). To further evaluate the PMI lipids, we utilized head group and FA composition to visualize structure–function relationships of the statistically significant species. Head group associations of all identified lipids were clustered by their structural similarity [34,40,42]. The resulting circular dendrogram is shown in Figure 3a with the adjusted p-value in the inner ring and log2FC on the outer ring. The most consistent observation relating to head groups was the upregulation of PC O-, PC P- and PE P-. The upregulation of SM lipids, another component of lipid bilayers abundantly present in lipid rafts and integral in cholesterol homeostasis, was also observed in the PMI study [58]. Conversely, PC lipids which have overlapping roles as charged species enriched within the outer lipid membrane layer were downregulated following a PMI [59]. Additionally, a general downregulation of glycerolipids was also detected following a PMI event, a contradictory finding to the positive correlation of TGs and MI incidence [60,61] This finding may instead reflect FAs serving as the primary energy substrates of the heart where non-esterified FAs, products of glycerolipids degradation, are rapidly complexed with CoA [62]. Notably, ceramides which have previously been positively correlated with cardiac disease risk were not observed to be statistically significant in our PMI cohort [63]. Exceptions to the head group trends, however, were noted for almost every class discussed herein. For example, we observed split dysregulation across SMs, CEs and other classes, illustrating effects beyond just head group influence. Figure 3. Lipids detected and statistically significant in the PMI comparison. (a) Of the 299 uniquely identified lipids, 141 were statistically downregulated and 66 were upregulated following a PMI with a p-value cut-off of 0.005. The lipid head group associations are visualized in a circular dendrogram with p-values (inner ring) and Log2FC (outer ring) statistics overlaid simultaneously for each lipid identification. (b) FA lipid composition was also investigated by plotting all unique FA components. Statistically significant lipids are shown in red and blue for up- and downregulation, and identified lipids lacking statistical significance are shown in grey. The magnitude of variation for Log2FC and adjusted p-values are visualized through a color gradient, with darker colors indicating a more significant p-value or larger fold change. Discrepancies between lipid head group composition and biological dysregulation suggest additional selectivity likely attributable to the FA components of lipid structures. Within FAs, important differences include chain length, and double bond number, position and orientation [64]. Previous efforts have elucidated FA chain length to directly influence cardiac pathology, but plasma studies have been less successful in capturing this effect [24]. To explore these associations, we further interrogated FA dysregulation in the identified lipids, as shown in Figure 3b. While the findings in these plots mainly correlated with the head group analyses, a few FA-specific observations could be extracted. First, a FA dependence of CE differential expression was observed—with 18 carbon-containing CEs being downregulated and CEs with 20 and 22 carbon PUFAs being upregulated. Long chain polyunsaturated fatty acids (LC-PUFAs) are a class of FAs characterized as having 18 or more carbons and at least two double bonds, often serving as precursors to lipid mediators [65]. PUFA dysregulation was also recognized among PE, PC and PE P- lipids; PE and PC lipids containing PUFA tails were downregulated, while the majority of significant PE P- lipids were upregulated. In an additional assessment of the summed FA double bond number, it was observed that while the majority of glycerolipids were statistically downregulated, the upregulated TG species had a greater number of unsaturation sites compared to the downregulated species. This is in agreement with models for predicting CVD onset, which have included unsaturated TG species [4,24]. However individual FA information was not attained for the majority of the TG species due to difficulties in assigning their MS/MS spectra. #### 3.2. Multi-Omics Results To assess how polar and nonpolar molecules change in both the ETT and PMI cohorts, the lipidomic results were integrated with the previously performed targeted analysis of 210 polar metabolites [26,27]. Results from these analyses elucidated both unique and shared statistically significant metabolites and biological processes across both events (Figure 4). For example, glycolysis and TCA cycle metabolites (red, pink and peach nodes) were upregulated following ETT, a finding agreeable with the known mechanisms of burning energy through high-intensity exercise [66]. Additionally, niacinamide, a component of NADH that is also associated with energy through its interaction with insulin, was found to be statistically upregulated with exercise. In PMI, the dietary metabolites of PC lipids previously shown to predict CVD risk, choline and trimethylamine N-oxide (TMAO), were downregulated and clustered next to each other to affirm their structural relationship [18]. Amino acid dysregulation was also observed following both ETT and PMI, as alanine was statistically significant through its upregulation immediately following exercise but downregulation following a PMI. Both ETT and PMI also shared an upregulation of xanthine and hypoxanthine, metabolites involved in purine metabolism and ATP degradation, which are notably upregulated following cellular damage. These xanthine metabolites can also interact with xanthine oxidase to produce reactive oxygen species, a mechanism well characterized in heart failure [67]. From our analysis comparing the lipidomic changes in ETT and PMI, we note unique profiles where plasma metabolite signals best characterized mechanistic changes following high-intensity exercise training. Conversely, we demonstrated an overwhelming dysregulation of lipids following a PMI in the end-stage phenotype of CVD, in addition to the metabolomic findings that were previously published. While the metabolomic analyses elucidated changes for both the ETT and PMI cohorts with slight overlap between each characterization, the lipidomics results were quite different. While no statistically significant lipids were noted in the ETT study, the sheer number of statistically significant lipid associations in the PMI cohort (207, 69% of identified lipids) provide striking evidence for the integral role of the lipidome immediately following a PMI event (Figure 5). The findings from the ETT cohort were, however, in accordance with other exercise studies which have observed lipidome disruption being proportional to the duration and intensity of exercise [68]. Previous characterizations of lipid variation in exercise have centered on the decrease in free carnitine and increase in short-chain acylcarnitine through its crucial capability of shuttling FAs into mitochondria within muscle tissue for energy usage [25,69]. Dysregulation of carnitines has faced some disagreement in literature, likely from the lack of correlation between muscle and plasma sampling [69]. Further, the energy sources of exercise differ substantially as low intensity training relies on fat as a primary fuel source, while high intensity training uses carbohydrates as an immediate energy supply [23]. From the observation of metabolite intermediates of glycolysis and the TCA cycle such as lactate being upregulated, we feel we can confidently state that known mechanisms of high intensity training were taking place in our cohort [26]. From the dysregulation of energy processes in the metabolomics data and the variation in carnitine species observed just above the significance cutoff, it is possible that these species were in fact perturbed in our system as has been noted by others (Supplemental Table S2) [52,69]. A variety of factors may preclude this annotation, including age-based impairment of the acyl carnitine pathway that diminishes FA oxidation and study-to-study variation from different exercise training regimes [54,70]. A lack of differential expression of the lipidome following exercise may also reflect that lipid variation is not always immediate [24]. The singular treadmill training event for this analysis, therefore, may be too short to assess any additional lipidomic changes [52]. From the known pathophysiology of over-exercise triggering the calcification of the heart muscle, the activation of lipid enzymes by Ca2+ may suggest more drastic lipidome dysregulation would be observed with repeated exercise training [24]. Figure 4. Statistically significant metabolites in the ETT and PMI studies. A circular dendrogram is utilized to showcase the differentially expressed metabolites in the PMI (inner ring) and ETT (outer ring) cohorts. Red and blue are used for up- and downregulation with a color gradient visualizing the magnitude of the adjusted p-value observed. Grey metabolites were detected but not statistically significant. Figure 5. The multi-omic assessment of statistically significant lipids and metabolites from a PMI event. Adjusted p-values for each molecule are shown around the dendrogram. Red and blue are used for up- and downregulation with a color gradient to visualize magnitude. From our analysis of 20 patients before and after a PMI event, we observed several instances of lipid dysregulation with substantial biological implications. Ether lipids (PC O-, PC P-, PE P-) have been shown to be disproportionately abundant in brain and heart tissues as components of the lipid bilayer with unknown biological implications [71]. While the biological significance of ether lipids overexpressed in heart tissue is not fully understood, the upregulation of these species in plasma following a PMI is likely indicative of tissue degradation following ASA treatment. However, only a subset of membrane lipids were upregulated, suggesting these lipid classes carry additional significance for ASA-induced PMI. This finding could potentially be explained by the preferential oxidation of O- and P- linkage sites that serve to protect the sn-2 FA group from oxidation [72]. In the heart, dysregulation of PC lipids in tandem with increased activity of phospholipase enzymes has been observed in CVD, where lysophospholipids contribute to atherosclerosis and vascular damage through their role in inflammation, as was observed here in their downregulation [73,74]. Altogether, the obstruction of heart tissue following a PMI could serve dual purposes, reflecting both a breakdown of ablated cellular tissue and dysregulation of essential biological processes, such as energy production and inflammation. PUFAs with a double bond on the third carbon (n-3) have previously been shown to serve preventative roles in CVD through their antiatherogenic effects and may explain the dysregulation of PUFA-containing lipids [75]. Metabolomic analysis of the PMI samples showed the most dysregulation among amino acids, where branched amino acids are precursors for glutamine and alanine synthesis in muscles [76]. Conversely, amino acids associated with cardiac remodeling (proline) were downregulated [77]. #### 3.3. Study Comparison Combining the two complementary stories of metabolite and lipid dysregulation before and after exercise and a PMI provides an important assessment of their biological changes (Figures 3 and 4). This comparison is incredibly insightful for understanding CVD pathophysiology, as shown by comparing our results with the general consensus of molecular dysregulation from several exercise, CVD onset and MI studies (Figure 6) [4,24,51,52,61,69,78]. Results from the CVD onset studies have illustrated upregulation of sphingolipids and carnitines, and shown downregulation of lyso PC and DGs. CVD onset metabolomic studies have also elucidated distinct molecular changes to include mechanisms of oxidative stress and PC degradation products promoting atherogenesis [55]. In the comparison of exercise, CVD onset and a PMI, there is quite substantial overlap in the perturbed processes, but the molecules being dysregulated are often unique. For example, different energy processes were dysregulated in both exercise and PMI, as glycerolipids were largely downregulated in PMI and TCA/glycolysis metabolites were upregulated with exercise. Differential expression of both 1-methyl histamine and lysophospholipids was also observed, and since both have been linked to roles in inflammation, this suggests a possible response to ASA treatment [73,74]. Uniquely, ether lipids, which were upregulated in PMI, are also recognized regulators of ion channels [71]. Relative to CVD onset, we noted a number of lipid and metabolite species dysregulated in both the ETT and PMI studies (Figure 6). For example, lysoPCs (LPC) were downregulated across PMI and are largely corroborated by literature [4,56]. The further annotation of choline and TMAO degradation products of PC lipids suggests an even greater significance in PC lipids for the development of end-stage perturbations, however the direction of change between CVD and the PMI model differed [55]. We also noted opposite trends when comparing CVD onset results from the literature and our ETT cohort; carnitines have been reported to be downregulated in exercise but upregulated in CVD onset, further reflecting the importance of the shift in energy processes between PMI and exercise [69,78]. These findings are significant for further elucidating the mechanisms of CVD, which we and others have shown reflect drastic changes in the lipidome but are missed from polar metabolomics experiments [4,48,51,53]. We would, however, like to note that the limited size of our patient study fails to capture sex-based differentiation of CVD onset established previously [79–82]. We also note limitations in our ETT analysis from a singular bout of exercise and disparities among patients from variables such as cardiovascular health history and ischemia that may hinder the elucidation of exercise-based lipid dysregulation. Figure 6. Comparison of lipidomic and metabolomic trends for exercise (left), cardiovascular disease (CVD) onset (middle) and planned myocardial infarction (PMI) model (right). Results include a summary of observed results from this and the referenced previous studies noted by citation number in the figure [4,24,51,52,61,69,78]. #### 4. Conclusions The metabolomic and lipidomic findings observed for the exercise and PMI cohorts showcased their unique pathophysiology. Of the statistically significant metabolites observed for both events, little overlap was found, implicating unique molecular processes for each [26,27]. Since the insights from a singular class of biomolecules are inherently limited, we expanded the metabolomic analyses to include lipids. Novel instrumentation platforms and cheminformatics tools were applied to provide confident lipid identifications and investigate lipid variation [38]. The lipidomic analyses illustrated how the exercise cohort had no statistically significant lipids after treadmill testing, while 69% of identified lipids were dysregulated one hour after a PMI. This finding was in itself very interesting and distinguished the molecular mechanisms for the two events. As such, the polar metabolites were more informative for the exercise study, while the lipidomic results provided a better assessment of the PMI cohort. Specifically, one hour following a PMI, lipid species with head groups including PC O-, PC P- and PE P- were all upregulated, while SMs were mainly upregulated and PCs were mostly downregulated. PUFAs were also selectively dysregulated across lipid head groups following a PMI. However, even with the lipid structural insight achieved, discrepancies in class trends were still observed, since LC–IMS–CID–MS analyses allow for the confident assignment of lipids, but analytical improvements are necessary to probe the roles of double bond position and orientation in these discrepancies. Interestingly, integrating the multi-omic exercise and PMI studies showed perturbation of energy processes across both events. The multi-omic analyses also corroborated findings from singular omic analyses where inflammation and atherogenic processes are heavily implicated in PMI. Furthermore, their comparison with CVD onset studies showed strong agreement between the lipid and metabolite dysregulation observed in the PMI cohort, and less agreement with the ETT cohort results, as expected. Ultimately, the integration of the lipid and metabolite data elucidated unique biological roles within molecular classes, providing complementary profiles for how preventative strategies and MI events greatly differ in their molecular mechanisms. Supplementary Materials: The following are available online at https://www.mdpi.com/2218-2 73X/11/1/40/s1, Table S1: ETT and PMI cohort information. Table S2: Exercise results. Table S3: PMI results. Author Contributions: M.T.O. and E.S.B. wrote the manuscript. K.G.S., L.M.B., B.-J.M.W.-R. and M.T.O. performed statistical analyses; and K.J.B., M.E.M. and E.S.B. performed the experiments. E.S.B. and K.E.B.-J. designed the experiments and supervised different aspects of the project. All authors have read and agreed to the published version of the manuscript. Funding: Portions of this research were supported by grants from the NIH National Institute of Environmental Health Sciences (P30ES025128, P42 ES027704 and P42ES031009) and startup funds from North Carolina State University. Institutional Review Board Statement: All blood sampling was performed as part of the human studies protocols approved by the Massachusetts General Hospital Institutional Review Board. Informed Consent Statement: Written informed consent was obtained from all subjects. All samples in this manuscript were also de-identified prior to the study. Data Availability Statement: Raw data is available through MassIVE (https://massive.ucsd.edu/ MSV000086620).R Code for recreating data visualization within this manuscript is available at https://github.com/BakerLabNCSU/PMI\_Exercise\_Multiomics. Acknowledgments: The data were collected in the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is a multiprogram national laboratory operated by Battelle for the Department of Energy (DOE) under Contract DE-AC05-76RLO 1830. Conflicts of Interest: The manuscript authors declare no conflict of interest. #### References ## Article Chronic High-Fat Diet Induces Early Barrett's Esophagus in Mice through Lipidome Remodeling Jeffrey Molendijk 1,2, Thi-My-Tam Nguyen 2, Ian Brown 3,4, Ahmed Mohamed 1, Yenkai Lim 2, Johanna Barclay 5, Mark P. Hodson 2,6,7, Thomas P. Hennessy 2,8, Lutz Krause 2, Mark Morrison <sup>2</sup> and Michelle M. Hill 1,2,\* Received: 1 April 2020; Accepted: 12 May 2020; Published: 16 May 2020 Abstract: Esophageal adenocarcinoma (EAC) incidence has been rapidly increasing, potentially associated with the prevalence of the risk factors gastroesophageal reflux disease (GERD), obesity, high-fat diet (HFD), and the precursor condition Barrett's esophagus (BE). EAC development occurs over several years, with stepwise changes of the squamous esophageal epithelium, through cardiac metaplasia, to BE, and then EAC. To establish the roles of GERD and HFD in initiating BE, we developed a dietary intervention model in C57/BL6 mice using experimental HFD and GERD (0.2% deoxycholic acid, DCA, in drinking water), and then analyzed the gastroesophageal junction tissue lipidome and microbiome to reveal potential mechanisms. Chronic (9 months) HFD alone induced esophageal inflammation and metaplasia, the first steps in BE/EAC pathogenesis. While 0.2% deoxycholic acid (DCA) alone had no effect on esophageal morphology, it synergized with HFD to increase inflammation severity and metaplasia length, potentially via increased microbiome diversity. Furthermore, we identify a tissue lipid signature for inflammation and metaplasia, which is characterized by elevated very-long-chain ceramides and reduced lysophospholipids. In summary, we report a non-transgenic mouse model, and a tissue lipid signature for early BE. Validation of the lipid signature in human patient cohorts could pave the way for specific dietary strategies to reduce the risk of BE in high-risk individuals. Keywords: lipid; lipidomics; cardiac metaplasia; Barrett's esophagus; esophageal adenocarcinoma; microbiota #### 1. Introduction There are two main forms of esophageal cancer: esophageal squamous cell carcinoma and esophageal adenocarcinoma (EAC) [1]. Over a period of three decades, the incidence of EAC has risen sixfold, while esophageal squamous cell carcinoma has remained relatively stable [2,3]. In the United States, incidence of EAC was estimated to increase from 0.40 to 2.58 cases per 100,000 between 1975 and 2009 [4]. From less than 5% of all esophageal cancer cases before the mid-1970s [5], EAC now represents almost half of all cases [2,3], making it one of the most rapidly increasing cancers in Western populations. Despite recent advances in surveillance and treatment protocols, the prognosis for patients with advanced EAC is poor, with a 5 year survival rate of less than 16%, and a median survival of less than 1 year [6,7]. EAC is widely accepted to develop via a stepwise sequence, as a consequence of gastroesophageal reflux disease (GERD). GERD leads to chronic inflammation in the esophagus and reflux esophagitis [8]. In ~10%–15% of GERD patients, the damaged squamous epithelium of the distal esophagus is replaced by cardiac mucosa with intestinal metaplasia, a condition termed Barrett's esophagus (BE) [9,10]. Although BE itself has limited adverse health effects, patients with BE have a 30–60-fold increased risk of developing EAC [11], with estimated annual progression rate of ~0.1%–0.5% per year [12,13]. In addition to GERD and BE, epidemiology studies have identified male gender, tobacco smoking and obesity as risk factors for EAC [14]. To investigate the causality and to delineate the molecular mechanisms of GERD, surgical rodent models have been reported [15], but with high mortality rates due to the challenging surgeries. An alternative approach using dietary intervention was reported by Quante et al. [16], using 0.2% deoxycholic acid (DCA) in drinking water as a mimic of GERD to induce Barrett's-like metaplasia in interleukin 1β transgenic mice. A follow-up study showed that high-fat diet (HFD) accelerated tumor development in the interleukin 1β transgenic mouse model [17]. While the authors report an increased inflammatory tumor microenvironment and altered intestinal microbiome as potential mechanisms, HFD may also promote EAC through lipid dyshomeostasis and esophageal dysbiosis. Circumstantial evidence suggests roles for both lipids and the esophageal microbiome in BE/EAC pathogenesis. Patients receiving cholesterol-lowering statin therapy exhibit reduced incidence of BE [18,19] and EAC [20–23]. Alterations to the esophageal microbiome have been reported in human esophageal tissues during BE/EAC disease progression [24,25], while gastric Helicobacter pylori infection, or altered gastric microbiota, may influence EAC development by modulating refluxate composition or frequency [26,27]. To evaluate the impact of obesity and/or GERD on esophageal tissue morphology, and to address the hypotheses that the pathogenic mechanisms of HFD or GERD involve esophageal microbiome and/or tissue lipids, we employed HFD dietary intervention and 0.2% DCA exposure in non-transgenic mice, to mimic obesity and GERD, respectively. The mouse model mimicking early BE was adapted from a previous report using BE transgenic mouse [16,17]. We found that a 9 month HFD increased esophageal tissue inflammation and cardiac metaplasia. DCA in drinking water increased the severity of HFD-induced esophageal inflammation and metaplasia segment length, potentially via increased esophageal microbiome diversity. Tissue lipidomics analyses revealed a phospholipid and sphingolipid signature associated with esophageal inflammation and cardia development. #### 2. Materials and Methods #### 2.1. Animal Experiments The study was approved by The University of Queensland Animal Ethics Committee. #### 2.1.1. Materials Chow diet (Irradiated Rat and Mouse Diet) and HFD (SF04-001) were obtained from Specialty Feeds (Western Australia). Both diets were produced as cylindrical pellets with a diameter of 12 mm and comparable fiber contents of 5.2% and 5.4% respectively. The standard chow provides 12% of digestible energy from fat, 23% from protein and 65% from carbohydrates, and contained 0.78% saturated fats, 2.06% monounsaturated fats and 1.88% polyunsaturated fats by weight. The HFD provides 43% of calories from fat, 21% from protein and 36% from carbohydrates, and contained 10.03% saturated fats, 8.24% monounsaturated fats and 5.11% polyunsaturated fats by weight. Both diets were wheat- and soy-based, but differed in the primary source of fat; namely, fish meal, mixed vegetable oils and canola oil for the standard chow, or lard and soybean oil for the HFD. Deoxycholate was obtained from Sigma (Missouri, USA). #### 2.1.2. Dietary Treatments Eight-week-old male C57BL/6 mice were randomly assigned to one of four treatment groups for 9 months (n = 12). Mice were housed in groups in autoclaved standard shoe-box cages in a ventilated rack system. Drinking water with or without deoxycholate was prepared and replaced fresh weekly. All interventions were performed during the light period of a 12 h/12 h light/dark cycle. #### 2.1.3. Tissue and Serum Collection Tissue was collected within the same 3 h window to avoid discrepancies due to circadian variations. Blood was collected via cardiac puncture under isoflurane anesthesia followed by cervical dislocation. Blood was centrifuged at 5000× g for 10 min at 4 ◦C, and serum removed and stored at –80 ◦C. Distal esophagus and gastroesophageal junction tissues were collected from each mouse. The entire gastroesophageal junction was fixed for histology, while distal esophageal tissues were cut in half lengthwise. One half was fixed in formalin for histology, and one half snap frozen in liquid nitrogen for 16S ribosomal DNA (rDNA) sequencing for microbiome analysis. #### 2.1.4. Histology Tissues were fixed in 10% formalin for 24 h and embedded in paraffin. Embedded tissue blocks were cut into 4 μm sections and used for hematoxylin and eosin (H&E) staining. Histological evaluation and grading was performed by a specialist gastrointestinal pathologist (IB). For grading, inflammation was graded on a scale of 0 to 3 (0 = nil inflammation; 1 = mild; 2 = moderate; and 3 = severe). The presence and length of cardiac-type mucosa was recorded. #### 2.2. Lipidomics Experiments #### 2.2.1. Materials SPLASH LipidoMix Mass Spec Standard mixture (#330707), containing deuterated lipids of 14 species at various concentrations, and the Ceramide/Sphingoid Internal Standard Mixture II (#LM-6005), were purchased from Avanti Polar Lipids, Inc. (Alabaster, U.S.A). ESI-L low concentration tuning mix (#G1969-85000) was purchased from Agilent Technologies (Mulgrave, VIC, Australia). #### 2.2.2. Lipid Extraction All steps except for sonication and sample blowdown were performed on ice. Serum and tissue samples were homogenized differently but lipids were extracted using the same methyl-tert-butyl ether (MTBE)/methanol extraction method [28]. Mouse serum (30 μL) was added to 215 μL of ice-cold methanol containing 50 μg/mL butylated hydroxytoluene (BHT). Samples were homogenized by three rounds of vortex mixing for 30 s, freezing in liquid nitrogen for 1 min, thawing for 2 min and sonicating for 10 min at 15 ◦C, power 100% in a Grant XUB18 bath sonicator. Tissue wet weight was determined using a Mettler-Toledo XS105 balance (Mettler-Toledo, Melbourne, Australia). Biopsies were transferred to Eppendorf tubes containing 500 μL ice-cold methanol, 50 μg/mL BHT and one steel bead and homogenized in a TissueLyzer LT (Qiagen, Melbourne, Australia) for six minutes at 50 Hz. Homogenate was transferred to new tubes and the original tube was washed with 400 μL methanol and transferred. Samples were dried down under nitrogen flow and resuspended in 20 μL water and 200 μL methanol (50 μg/mL BHT). Samples were homogenized by three rounds of vortex mixing for 30 s, freezing in liquid nitrogen for 1 min, thawing for 2 min and sonicating for 10 min at 15 ◦C, power 100% in a Grant XUB18 bath sonicator. SPLASH LipidoMix Mass Spec Standard (10 μL) and Cer/Sph mixture II (10 μL) internal standards mixes from Avanti Polar Lipids were then added to each sample. After overnight incubation at −30 ◦C, 750 μL MTBE was added and each tube was vortex mixed for 10 s and shaken for 10 min on a tube rotator (4 ◦C). MilliQ water (188 μL) was then added, and the tube was vortex mixed for 30 s to form a biphasic separation. After centrifuging for 15 min at 15,000× g, 700 μL of the clear upper phase containing lipids in MTBE was transferred to another tube and dried down using a gentle stream of nitrogen. After drying down of lipids, extracts were resuspended in 50 μL methanol (containing 50 μg/mL BHT)/toluene (90%/10%, v/v). Dry weight of the remaining pellets from tissue samples was determined in triplicate using a Mettler-Toledo XS105 balance. Dry weights were used to normalize lipid injection volumes of tissue samples prior to mass spectrometry analysis. For serum samples equal volumes were injected. #### 2.2.3. Untargeted Lipidomics An Agilent Technologies 1290 Infinity II UHPLC system with an Agilent ZORBAX Eclipse plus C18 1.8-micron column (#959757-902) and guard column (#821725-901), coupled online to an Agilent 6550A iFunnel QTOF mass spectrometry system, was used for untargeted lipidomics. The mass spectrometer was tuned in the low mass range (1700 m/z), high sensitivity slicer mode and the instrument mode was set to Extended Dynamic Range (2 GHz). The quadrupole and time-of-flight (TOF) sections of the mass spectrometer were both tuned prior to each experiment. The quadrupole was tuned to reference masses 118.09, 622.03 and 1221.99 in positive ionization mode. Experiments were performed if the quadrupole component passed the check tune for each reference mass in wide, medium and narrow modes. The TOF component was tuned using reference masses 118.09, 322.05, 622.03, 922.00, 1221.99 and 1521.97 in positive ionization mode. TOF mass calibration indicated that at around 110–120 m/z the resolution was ~12,000–13,000 and increased to 20,000–21,000 around 600–620 m/z range. The ion source used was Dual Agilent Jet Stream electrospray ionization, which allows for the simultaneous introduction of sample and reference masses into the mass spectrometer. Source capillary voltages were set to 4000 V for positive ionization mode whilst the nozzle voltage was set to 0 V, fragmentor was set to 365 and octopoleRFPeak to 750. Nitrogen gas temperature was set to 250 ◦C at a flow of 15 L/minute and a sheath gas temperature of 400 ◦C at a flow of 12 L/min. During the experiment reference masses were enabled (121.05 and 922.01 Da) to enable auto-recalibration of compounds with known masses. MS1 data was acquired between 100–1700 m/z at a scan rate of 2.5 spectra per second. The sample dilution and injection volume used for experiments was determined by testing a representative sample prior to analyzing the cohort. Reversed phase buffers A and B contained 25 millimolar (mM) ammonium formate and 0.1% formic acid in 60%/40% (v/v) acetonitrile/water or 90%/10% (v/v) isopropanol/water respectively. The separation gradient was run at a flow rate of 0.5 mL/min to separate the lipids during a 16 min gradient. The method started at 15% B and increased to 30% B at 2:00, 48% B at 2:30, 82% B at 11:00, 99% B at 11:30. The gradient was retained at 99% B until 13:00 and retained at the starting condition of 15% B between 13:06 and 16:00. The column compartment was maintained at 60 ◦C for the duration of the experiment. #### 2.2.4. Targeted Lipidomics Targeted lipidomics were performed on an Agilent Technologies 1290 Infinity UHPLC system with an Agilent HILIC Plus RRHD 2.1 × 100 mm 1.8 micron column, coupled online to an Agilent 6490A Triple Quadrupole mass spectrometer with iFunnel and Agilent Jet Stream electrospray ionization source, operated in dynamic MRM mode. The source nitrogen gas temperature was set to 250 ◦C at a flow rate of 15 L/min, and the sheath gas temperature set to 400 ◦C at a flow rate of 12 L/min. The capillary voltage was set to 4000 V for positive mode and 5000 V for negative mode and the nebulizer operated at 30 psi. Ion funnel low and high pressure in positive mode were 150 and 60, and in negative mode 150 and 120, respectively. Check tunes were performed in wide, unit and enhanced modes prior to each experiment to confirm the performance of the mass spectrometer. The quadrupole was tuned to reference masses 118.09, 322.05, 622.03, 922.01 and 1221.99 in positive ionization mode, and 112.99, 302.00, 601.98, 1033.99 and 1333.97 in negative ionization mode. Each sample was analyzed in 3 separate dynamic MRM runs using two different HILIC buffer systems, both using 50%/50% (v/v) acetonitrile/water as Buffer A and 95% acetonitrile/water (v/v) as buffer B. The buffers were supplemented with 25 mM ammonium formate, pH 4.6 and 0.1% formic acid (denoted methods F1, F2) or 10 mM ammonium acetate, pH 7.6 (denoted method A). As detailed in Table S1, the methods had 155 (F1), 156 (F2) and 126 (A) transitions, including internal standards. The minimum dwell times were 4.2 milliseconds (ms), 4.1 ms and 3.1 ms respectively for methods F1, F2 and A. The method started at 0.1% A and increased to 40% A at 8:00, 90% A at 9:30 until 10:30. The gradient decreased to 0.1% A between 10:30 and 11:30 and was retained at the starting conditions of 0.1% A until 14:00. The column compartment was maintained at 30 ◦C for the duration of the experiment. A pooled quality control (QC) sample was injected multiple times to condition the HPLC column prior to analyzing samples, and also queued after every 6–7 biological samples to monitor mass spectrometry performance for the duration of the experiment [29,30]. #### 2.2.5. Data Treatment and Analysis Feature integration of untargeted lipidomics data was performed using the XCMS Centwave method and retention time alignment was performed using the Obiwarp method [31]. Features were grouped and peak filling was performed using the fill ChromPeaks method. Finally, feature information and abundances per samples were exported as a .csv file format. Lipid identification was performed using MS-DIAL version 3.90 (RIKEN Center for Sustainable Resource Science, Kanagawa, Japan) and the included FiehnRT (v47) lipid database [32]. Identifications were made based on accurate mass, retention time and database matching, and then manually confirmed. The MS1 tolerance was set to 0.01 Da and the tolerance for MS2 peaks was set to 0.05 Da. Database retention times were not used for scoring in the lipid identification. An identification score cut-off of 70 was set to remove most inaccurate identifications. The possible adduct ions were set to [M + H]+, [M + NH4] <sup>+</sup> and [M-H]−. Manual confirmation included the visual inspection of all database matches, assessing the dot and reverse dot product similarity scores. Ambiguous identifications of features with multiple likely identifications were excluded from the analysis. Lipid identifications, accurate masses and retention times were exported from MS-DIAL and integrated into the data exported from XCMS. Acquired targeted lipidomics data was imported into Skyline (MacCoss Lab, Department of Genome Sciences, University of Washington) [33], peak integration was automated but manually confirmed and corrected if required. Internal standard retention time was used to confirm correct peak integration of lipids belonging to the same class. Peak areas were exported from Skyline for further analysis in R (R Foundation for Statistical Computing, Vienna, Austria) [34]. The datasets were filtered to remove any lipids with a coefficient of variation greater than 20% among the quality control samples. Missing values were imputed using the MinDet method from the imputeLCMD R package using the default q-value of 0.01. All datasets were log2 transformed and normalized using the probabilistic quotient normalization method as described by Dieterle et al. [35]. Lipid information such as lipid class, number of unsaturated bonds and fatty acid chain lengths were parsed from the original lipid names using the lipidr R package [36]. Further analyses and visualizations, including principal component analysis (PCA) and lipid class boxplots were produced using lipidr [36]. The enrichment of lipid classes was determined using the LSEA (lipid set enrichment analysis method) [36]. Pearson correlation was used to determine the correlation between total lipid fatty acid chain lengths and the development of disease conditions. #### 2.3. Microbiome Profiling #### 2.3.1. DNA Extraction Unless otherwise stated, solvents were purchased from Sigma (Missouri, USA). Mouse tissues were preincubated with lysis buffer (20 nanomolar (nM) Tris/HCl; 2 mM EDTA; 1% Triton X-100; pH 8; supplemented with 20 mg/mL lysozyme) for 60 min at 37 ◦C, then with 25 μL Proteinase K (20 mg/mL; Ambion, CA, USA) at 56 ◦C until completely lysed. DNA was extracted using the ISOLATE II Genomic DNA Kit (Bioline, London, UK) following manufacturer's standard protocol. The DNA samples were eluted in two lots of 50 μL Elution Buffer G from the kit. #### 2.3.2. Library Preparation and Sequencing Library preparation was performed in batch. Polymerase chain reaction (PCR) preparation was conducted in a designated DNA template-free room. Sequencing library preparation of the samples and control (no DNA template) was based on the 16S Metagenomic Sequencing Library Preparation guidelines provided by Illumina. Q5 Hot Start High-Fidelity 2× Mastermix polymerase (NEB, Ipswich, MA, USA) was used for the Amplicon PCR step. Primers used for the amplification of the V6–V8 region of the 16S ribosomal RNA gene were primers 927-Forward (AAACTYAAAKGAATTGRCGG; universal) and 1392-Reverse (ACGGGCGGTG WGTRC; universal) with Illumina adapter sequences. Samples were barcoded using the Illumina dual-index system (Nextera XT v2 Index Kit Set A) for the Index PCR step. PCR products were purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). The DNA concentration for each barcoded amplicon mixture was quantified following manufacturer's instructions (Quantus, Promega, Madison, WI, USA) and all samples were pooled to provide 4 nanomol of each amplicon. The pooled libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and the MiSeq Reagent Kit v3 (2 × 300 bp) by the Australian Centre for Ecogenomics, located at the University of Queensland. #### 2.3.3. Bioinformatics and Statistical Analysis Raw sequencing reads were processed and analyzed using Quantitative Insights Into Microbial Ecology 2 (QIIME 2, version 2019.7) according to the developer's recommendations [37]. Sequence quality control was carried out using the DADA2 algorithm, a QIIME 2 plugin-software to filter low-quality sequences as well as to identify and remove chimeric sequences. Amplicon sequence variants (ASVs) were generated from the filtered sequences and the SILVA\_132 99% reference database was used to train the feature classifiers and provide taxonomic assignment accordingly. An ASV table was generated and normalized using total sum normalization (TSS) for all further analyses using Calypso (version 8.84) [38]. #### 3. Results #### 3.1. High-Fat Diet and Bile Acid Exposure as a Mouse Model for the Development of Esophageal Inflammation and Cardiac Metaplasia Chronic treatment with the unconjugated bile acid, deoxycholic acid (DCA, 0.2%), in drinking water was previously reported to accelerate Barrett's-like metaplasia development in an interleukin-1β transgenic mouse model [16]. We hypothesized that obesity induced by chronic HFD will replicate the chronic inflammation due to interleukin-1β overexpression, and leads to Barrett's-like epithelium development in wild-type mice. To test this hypothesis, male C57BL/6 mice were fed with standard chow diet or HFD with and without 0.2% DCA, for 9 months prior to sacrifice (n = 11 per group). Chow and HFD diets had comparable fiber (5.2% vs 5.4%) and protein (23% vs 21%) content, but the digestible energy from fat increased from 12% in chow to 43% in HFD, while carbohydrate reduced from 65% to 36%. Body weight was monitored weekly, and HFD +/− DCA mice had significantly higher body weight than Chow +/− DCA (q < 0.0001), but no difference in body weight was observed between mice +/− DCA in either diet group (Figure 1a). Interestingly, weight gain in mice in the HFD + DCA group was delayed compared to the HFD + water group, potentially indicative of DCA-induced esophageal damage reducing food intake and subsequent recovery (Figure 1a). Figure 1. Chronic high-fat and/or bile acid dietary intervention in wild-type mice induces chronic inflammation and cardiac metaplasia development at the gastroesophageal junction. C57BL/6 mice (n = 11 per group) were given +/− high-fat diet (HFD) and +/− 0.2% deoxycholic acid (DCA) over a 9 month period, and gastroesophageal junction tissue morphology evaluated in hematoxylin & eosin (H&E) stained sections for inflammation and epithelial changes. (a) Body weight over time for each of the four groups. Values are mean ± SD; (b) Example esophageal epithelium morphology for normal and cardiac metaplasia. (c) Example inflammation grading. (200×; scale bar 100 μm). Next, we examined the impact of the dietary treatments on tissue morphology of the gastroesophageal junction, where BE arises. H&E stained tissues were evaluated, and graded for inflammation severity and metaplasia length by an expert gastrointestinal pathologist in a blinded manner. Figure 1b shows the morphology of the normal squamous epithelium of the gastroesophageal junction, which was observed in most samples. In contrast, cardiac metaplasia with neutral mucin-producing glands was observed immediately adjacent to the squamous epithelium, observed in all four groups with varying frequency. Furthermore, varying grades of inflamed esophageal tissue were observed (Figure 1c). Inflammation grade 0 lacks inflammatory cells in the lamina propria, whereas mild inflammation with small numbers of lymphocytes and eosinophils are observed in inflammation grade 1. Inflammation grade 2 is marked by moderate inflammation, with a prominent infiltration of the lamina propria by lymphocytes and small numbers of eosinophils. Additionally, lymphocytes infiltrate the squamous epithelium. In severe inflammation, grade 3, a prominent infiltration of the lamina propria by lymphocytes, plasma cells, eosinophils and neutrophils is observed. Neutrophils and eosinophils are present within the epithelium. Quantitative analysis revealed a basal level of mild inflammation in ~20% of the control and DCA treatment groups (Figure 2a). The combined HFD + DCA increased the overall incidence of inflammation to 67%, and was the only group with a grade of severe inflammation (Figure 2a). HFD alone slightly increased inflammation incidence to 27%, but induced a similarly high level of metaplasia (64%–67%) as the combined HFD + DCA (Figure 2b). However, all of the instances of metaplasia for the HFD + DCA group were long segment, while metaplasia induced by HFD alone comprised short, medium and long segments (Table S2). Figure 2. Synergistic action of chronic HFD and DCA promotes inflammation and cardiac metaplasia at the gastroesophageal junction. H&E stained tissue sections graded for the degree of inflammation (mild, moderate or severe), and the length of cardiac metaplasia (short, medium or long) were analyzed for (a) the occurrence and degree of inflammation, and (b) length of cardiac metaplasia in the four treatment groups. Correlation between presence of cardiac metaplasia was further compared for: (c) all mice treated with DCA compared to water control; (d) all mice on HFD diet compared to chow diet; and (e) any level of inflammation. The significance for plots c–e was calculated using the Fisher's exact test. \\ p-value < 0.05. The above results demonstrate that chronic HFD with DCA (mimicking GERD) induces the hallmarks of early BE, namely, tissue inflammation and metaplasia. To further evaluate the correlation between each dietary treatment, we next asked if the inflammation or metaplasia incidence correlate with HFD or DCA treatment. When all samples from DCA treatment groups were compared against all groups treated with water, no significant difference was detected for incidence of cardiac metaplasia (Figure 2c). Similarly, HFD, with or without DCA, did not significantly increase the development of cardiac metaplasia (Figure 2d). Finally, we asked whether the incidence of inflammation and metaplasia was correlated, and found a significant relationship, with 6% of mice without esophageal inflammation and 54% of mice with inflammation developing cardiac metaplasia (Figure 2e). Furthermore, among the mice that developed cardiac metaplasia, the mice with inflammation developed a longer metaplastic tissue (Figure 2e). #### 3.2. Esophageal Tissue Microbiome Diversity Increases with DCA After confirming the induction of gastroesophageal junction inflammation and cardiac metaplasia by chronic HFD + DCA treatment, we went on to profile the esophageal microbiota of 43 samples from the four study groups, using 16S ribosomal RNA gene sequencing. One sample gave no sequences and was removed from subsequent analysis. In total, 21,708 high quality sequences were obtained, with an average of 504.84 sequences per sample. From these sequences, four major phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) were identified, and a total of 23 ASVs were detected at 99% sequence identity threshold via SILVA\_132 database. We first compared microbial diversity (Shannon index) and richness between treatment groups using rank test in Calypso. No significant differences in microbial richness was observed, but a higher microbial diversity was observed in DCA alone, and HFD + DCA groups (Figure 3a). To further test the relationship between DCA and microbial diversity, we then re-grouped the data into HFD-treated and DCA-treated groups, as previously done (Figure 2). While no significant differences in microbial diversity or richness were detected for HFD treatment (Figure 3b), a significant increase of microbial diversity in DCA-treated groups was detected, with a similar but non-significant increase in richness (Figure 3c). Figure 3. Esophageal microbiome diversity is increased by HFD + DCA treatment. Shannon index and microbial richness of esophageal microbiome data was measured using rank test for (a) each of the four treatment groups, (b) combining HFD/Water and HFD/DCA groups into the HFD group, and Chow/Water + Chow/DCA into the Chow group, or (c) combining Chow/DCA and HFD/DCA groups into the DCA group, and Chow/Water + Chow/DCA into the Water group. \* p-value < 0.01. #### 3.3. Lipidomic Changes Associated with Dietary Interventions In parallel to the esophageal microbiome analysis, we conducted lipidomics analyses on the collected serum and gastroesophageal junction samples, to determine associations between the respective lipidomes and dietary treatments (HFD or DCA), inflammation or cardiac metaplasia. A combined approach of untargeted and targeted lipidomics was conducted, to quantitate 339 and 197 mammalian lipid species in the serum and gastroesophageal junction samples, respectively. While we observed no separation of gastroesophageal junction lipidome as a result of dietary treatments by PCA in the first two principal components (Figure 4b), the serum lipidome showed clear separation and clustering according to dietary intervention groups (Figure 4a). Differential expression analysis was conducted on the lipidomics data of both datasets. Lipid class enrichment was conducted to determine if specific lipid classes were selectively altered. The boxplots in Figure 4c,d summarize the log2 fold change for each lipid class for each group, for serum and gastroesophageal junction tissue lipids, respectively. Statistically significant changes are colored in blue. Gastroesophageal junction tissue lipid class analysis (Figure 4d) revealed overlapping impacts of HFD and DCA treatments. All three treatment groups showed elevated lysophosphatidylcholine (LPC), as well as decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (Figure 4d). While triacylglycerol (TAG) was elevated only in group B (DCA alone), phosphatidylglycerol (PG) was elevated in HFD-treated groups (Figure 4d). For serum lipids, both HFD-treated groups (C and D) show similar changes, with elevated ceramide (Cer), PG and sphingomyelin (SM), and reduced lysophosphatidylethanolamine (LPE), PE and phosphatidylinositol (PI) (Figure 4c). In contrast, DCA treatment alone (Group B) showed a large decrease in ether-PC, with modest changes in PI and SM (Figure 4c). Interestingly, the reduction in ether-PC was not observed in the combined HFD + DCA treatment (Group D), suggesting HFD rather than DCA is the main driver of the serum lipidome. #### 3.4. Lipidomic Changes Associated with Early Tissue Pathology Since esophageal inflammation or metaplasia occurred in ~10% to 70% of mice in each group, we next investigated the association between serum and gastroesophageal junction tissue lipidome with early esophageal pathology. To this end, lipid class enrichment analysis was conducted on metaplasia vs normal samples, and inflamed vs normal samples. Apart from elevated serum ether-PC, the serum lipidome returned minor changes of < 25% magnitude (Figure 5a). In contrast, the tissue lipidome showed similar changes for metaplasia and inflammation, characterized by reduced lysolipids and elevated ceramides (Figure 5b). This result revealed major differences between the lipidome associated with dietary intervention (Figure 4) and that associated with esophageal pathology (Figure 5). Specifically, differences were observed for ceramides and the lysolipids LPC and LPE. Elevated tissue ceramide was associated with metaplasia and inflamed tissue, but not with any dietary treatment, even in the HFD + DCA treatment group, where 66.7% of cases were inflamed (Figure 5). Reductions in the lysolipids LPC and LPE were associated with metaplasia and inflammation (Figure 5b), but elevated tissue LPC was associated with HFD and DCA treatment (Figure 4d). These results strongly implicate roles for elevated ceramides and reduced lysolipids in metaplasia development due to chronic inflammation. Figure 4. The impact of dietary interventions on tissue and serum lipidome. After 9 months high fat diet (HFD) +/- 0.2% deoxycholate (DCA), mouse gastroesophageal junction tissue and serum samples were subjected to lipidomics analyses. (a) Principal component analysis score plot of mean-centered unit variance-scaled untargeted serum lipidome data (n = 38). (b) Principal component analysis score plot of mean-centered unit variance-scaled untargeted gastroesophageal junction tissue lipidome data (n = 29). Plot ellipses represents the 95% Hotelling's T2 confidence intervals for the multivariate data. (c,d) Lipid class boxplots for serum and gastroesophageal junction tissue lipids, showing the distribution of log2 differences between the treatment group and control. Positive values represent lipids that are more abundant in the treatment group than in the control group. Blue color indicates significant enrichment using the fast gene set enrichment analysis (fgsea) method. Cer—Ceramide, LPC—lysophosphatidylcholine, LPE—lysophosphatidylethanolamine, PC—phosphatidylcholine, PE—phosphatidylethanolamine, PG—phosphatidylglycerol, PI—phosphatidylinositol, SM sphingomyelin, DAG—diacylglycerol, TAG—triacylglycerol. Figure 5. Lipid classes associated with gastroesophageal junction tissue pathology. (a,b) lipid class boxplots for serum and tissue lipids, showing the distribution of log2 differences between the disease condition and control. The disease conditions cardia and inflammation were visualized after applying the removeBatchEffect function from the limma R package. Positive values represent lipids that are more abundant in the disease condition group than in the control group. Blue color indicates significant enrichment using the fast gene set enrichment analysis (fgsea) method. Cer—Ceramide, LPC—lysophosphatidylcholine, LPE—lysophosphatidylethanolamine, PC—phosphatidylcholine, PE—phosphatidylethanolamine, PG—phosphatidylglycerol, PI—phosphatidylinositol, SM—sphingomyelin, DAG—diacylglycerol, TAG—triacylglycerol. As differing fatty acid chain lengths on a lipid can greatly impact biological function in cancer development [39], we next determined whether fatty acid chain lengths were associated with inflammation or metaplasia for the Cer, LPC and LPE classes. Ceramides comprise a single fatty acid chain with a sphingoid backbone (commonly 18:1, as illustrated in Figure 6a). Figure 6a plots the log2 fold change for different total fatty acid chain lengths of each measured ceramide species. As evident in Figure 6a, a significant correlation was found between very long chain ceramides and the disease conditions inflammation and metaplasia. On the other hand, specificity in fatty acid chain lengths were not observed for LPC in either metaplasia or inflamed tissues (Figure 6b). Increased LPE chain lengths were significantly correlated with metaplasia, but not with inflammation (Figure 6c). Figure 6. Association of tissue ceramide, LPC and LPE fatty acid chain lengths with gastroesophageal junction tissue pathology. (a–c) Total chain length plots for tissue lipids, showing the alterations in log2 abundances between the disease condition and control. The x-axis labels refer to the total fatty acid chain length of the measured lipid. The disease conditions metaplasia and inflammation were visualized after applying the removeBatchEffect function from the limma R package. Positive values represent lipids that are more abundant in the disease condition group than in the control group. The metrics shown in the plots refer to the Pearson correlation coefficient (R) and p-value. The smoothed line and 95% confidence interval were drawn using geom\_smooth, by fitting a linear model. The structures above each plot represent lipid species of the Ceramide, LPC and LPE classes, where the R groups refer to the hydrocarbon chains of varying lengths. #### 4. Discussion This is the first study to demonstrate that chronic HFD in non-transgenic mice is sufficient to induce esophageal inflammation and cardiac metaplasia, the first steps in BE/EAC pathogenesis. While DCA in drinking water had no effect on esophageal morphology on its own, it increased the severity of inflammation and length of metaplasia when combined with HFD. HFD clearly induced obesity and serum lipid derangements, but only a proportion of HFD-treated mice developed esophageal inflammation and cardiac metaplasia. Intriguingly, the esophageal tissue lipidome showed a similar signature for inflammation and metaplasia, which was not associated with HFD. These results suggest that homeostatic mechanisms can buffer HFD/obesity-induced lipidome derangement to an extent, beyond which inflammation and metaplasia ensue. Obesity increases the risk of several cancer types, and the mechanisms of specific lipids on carcinogenesis are beginning to be revealed [39]. In this study, we identified an esophageal tissue lipid signature for inflammation and metaplasia, which is characterized by elevated very long chain ceramides and reduced lysolipids, LPC and LPE. Very long chain ceramides have been reported to increase cancer proliferation, and evade growth suppressor and apoptotic signals [39]. A link between HFD and tissue ceramide levels was recently reported by Zalewska et al. [40] for submandibular gland ceramide following HFD treatment in mice. The authors suggested that elevated ceramide increased mitochondrial reactive oxygen species (ROS) production and respiratory chain, leading to inflammation [40]. Phospholipid remodeling has recently emerged as playing an important role in disease pathogenesis, through the characterization of the lysophosphatidylcholine acyltransferase (LPCAT) family [41]. Lysolipids LPC and LPE contain a single fatty acyl chain, while the more abundant PC and PE contain two fatty acyl chains. Due to the differing biophysical properties, altered lysolipid:phospholipid ratio can lead to altered membrane curvature and fluidity, which could translate to organelle remodeling and altered signal transduction in pathology [41]. Warnecke-Eberz et al. [42] identified the LPCAT1 gene to be elevated in late- and early-stage esophageal adenocarcinoma tissue, compared to adjacent normal tissue. Elevated LPCAT1 could explain the decreased LPC and increased PC that we identified for inflamed and cardia gastroesophageal junction tissue (Figure 5). LPCAT1 enzyme and LPC are elevated in several other cancers, including colorectal cancer [43], hepatocellular carcinoma [44], gastric cancer [45] and clear cell renal carcinoma [46]. Interestingly, body fatness is a risk factor for all these cancers [47]. In a recent study of western diet-associated non-alcoholic steatohepatitis, LPCAT1 and LPCAT2 are in the top 10 liver genes/transcripts most significantly elevated in mice fed western style diets compared to standard diets [48]. Together, these data suggest a mechanistic link between high-fat diet, activation of LPCAT transcripts, altered LPC:PC ratio, and induction of esophageal inflammation and metaplasia development. As GERD is a well-established risk factor for BE, the lack of esophageal pathology from the mice treated with DCA alone was somewhat surprising. This result may suggest that 0.2% DCA in drinking water does not fully mimic GERD, or that GERD is less damaging to mice esophagus compared to human. Nevertheless, as expected for the additive effect of risk factors, DCA treatment in addition to HFD increased the severity of inflammation and length of metaplasia, compared to HFD treatment alone. DCA treatment increased the esophageal microbiome diversity, which is consistent with previous reports describing the effect that levels of bile acids in the gut have on the major division/phyla level taxa of the gut microbiome [49]. These effects could potentially extend to the esophagus, given that the composition of the esophageal microbiome depends on the oral and gut microbiome [50]. Previous studies have reported a depletion of Gram-positive Streptococcus, and enrichment of Gram-negative taxa, including Veillonella and Prevotella, in BE [25,51]. Interestingly, dysplasia and esophageal adenocarcinoma were reported to have reduced esophageal microbiome diversity [24]. Further studies will be required to establish the cause–effect relationship and mechanisms of esophageal microbiota in BE/EAC pathogenesis. #### 5. Conclusions In conclusion, we report the results of a dietary intervention model for early BE, and a lipidomic signature for inflamed and metaplastic esophageal tissue. In non-transgenic mice, chronic HFD was sufficient to induce inflammation and cardiac metaplasia at the gastroesophageal junction. As a GERD-mimic, bile acid in drinking water in addition to HFD increased the severity of inflammation and length of metaplasia. GERD, but not HFD, increased the esophageal microbiome diversity. The causality of microbiome in BE development remains to be established. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/5/776/s1, Table S1: Targeted lipidomics methods. Table S2: Tissue pathology grades. Tissue and serum lipidomics data are available on Metabolomics Workbench with Study IDs ST001323 and ST001336. Author Contributions: Conceptualization, J.B. and M.M.H.; methodology, J.M., L.K., M.P.H. and M.M.; formal analysis, J.M., A.M., Y.L., I.B.; investigation, J.M., T.-M.-T.N. and J.B.; resources, T.P.H., M.M.H.; writing—original draft preparation, J.M. and M.M.H.; writing—review and editing, T.-M.-T.N., A.M., L.K., Y.L., M.P.H., T.P.H.; supervision, M.M., M.P.H., T.P.H. and M.M.H.; funding acquisition, J.B., T.P.H. and M.M.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Translational Research Institute Spore Grant to M.M.H., J.B. and T.P.H. J.M. and T.-M.-T.N. were supported by Australian Postgraduate Research Awards. Metabolomics Australia is part of the Bioplatforms Australia network, funded through the Australian Government's National Collaborative Research Infrastructure Strategy (NCRIS). Translational Research Institute is supported by a grant from the Australian Government. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. #### References © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). ## Article Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture Laura Niederstaetter 1,†, Benjamin Neuditschko 1,2,†, Julia Brunmair 1, Lukas Janker 1, Andrea Bileck 1,3, Giorgia Del Favero 4,5 and Christopher Gerner 1,3,\* Abstract: Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments. Keywords: batch variations; eicosanoids; fetal calf serum; mass spectrometry; peroxisomes; proteomics #### 1. Introduction Problems with the inter-laboratory reproducibility of results obtained with in vitro cell culture models are increasingly being recognized [1,2]. The need to reduce the use of animal models for research purposes relies also on the use of accurate in vitro test models [3,4]. Important decisions such as the choice of drug candidates to be evaluated in clinical studies may be based on such experiments [5]. Thus, the identification of influencing factors potentially modulating such in vitro data is mandatory. Biological materials and reference materials have been recognized as the main contributors for irreproducibility, resulting in a current focus on the investigation of genetic heterogeneity and genetic instability of cell culture models [6,7]. Here we present Fetal Bovine Serum (FBS; also fetal calf serum, FCS) as another relevant contributor to reproducibility issues. FCS is commonly used as cell culture supplement sustaining the growth and duplication of mammalian cells in vitro. Citation: Niederstaetter, L.; Neuditschko, B.; Brunmair, J.; Janker, L.; Bileck, A.; Del Favero, G.; Gerner, C. Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture. Biomolecules 2021, 11, 113. https:// doi.org/10.3390/biom11010113 Received: 9 December 2020 Accepted: 14 January 2021 Published: 15 January 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Since its introduction in the 1950s its use has been established world-wide, irrespective of evident limitations regarding scientific as well as ethical points of view [8]. Fetal serum is basically a by-product of meat production collected from the still beating hearts of living fetuses. While efforts are made to reduce the use of FCS, they have shown rather limited successes [9]. As for other supplements of natural origin, the main variability source associated to FBS can be traced back to largely uncharacterized bioactive components. Due to low concentrations or lack of experimental standard measurements, they may remain poorly controlled, but may still influence the outcome of cell-based experiments. While some effort has been spent to define the composition of FCS, the main bioactive constituents subjected to meaningful variation are hardly known [8]. Batch-dependent variations have been described to affect biological outcomes but such considerations remain limited to rather specialized topics such as hormone regulation [10]. Chemically defined media (CDM) represent a general and consequent solution for these problems, but have only been established and available for a limited number of cell model systems [9]. The focus of the present study was to investigate whether it was possible to identify bioactive compounds in FCS accounting for relevant batch-specific effects and correlate this information with proven and biochemically evident readouts on cell functions. Variations of amino acid and metabolite composition of FCS may be considered as less likely as they should be subjected to the homoeostatic control of the organism and, further limited by the dilution of FCS in the accurately produced cell culture media also containing these molecules (typically 5–10% FCS is used). Thus proteins as well as eicosanoids and other polyunsaturated fatty acids (PUFAs) are profiled as the most relevant bioactive candidate molecules to account for inter-batch variation. Indeed, they are responsible for regulating biological processes associated with inflammation [11] and inflammation-associated pathomechanisms [12–14]. The monocyte cell line U937, a well-established cell model for macrophages [15–17], was chosen for these investigations. Overall, the data demonstrated significant effects of FCS-contained eicosanoids with batch-dependent variations on relevant cell functions, proving that bioactive lipid content in serum contributes to reproducibility issues in cell culture experiments. #### 2. Results Formation of bioactive pro-inflammatory mediators by macrophages may be influenced by FCS batch effects. In order to systematically investigate cell culture reproducibility issues resulting from FCS batch effects, a proteome profiling experiment using a U937 macrophage differentiation and activation model was performed. A single batch of U937 cells was seeded into 24 identical aliquots, forming four groups subsequently sub-cultured with four different FCS batches (Table 1, see Materials and Methods). All cells were differentiated using phorbol 12-myristate 13-acetate (PMA) to induce macrophage formation as verified by FACS (fluorescence activated cell sorting) analysis (Supplementary Figure S1), while three aliquots of each group (FCS batch) were subsequently treated with lipopolysaccharides (LPS) to induce inflammatory stimulation, the other three per group serving as untreated controls. The formation of inflammatory mediators was investigated by comparative secretome analysis resulting in the identification of 488 proteins (Supplementary Table S3) and 54 eicosanoids and fatty acid precursor molecules (Supplementary Table S4). Whereas most molecules such as the chemokine CCL3 and CXCL5 showed rather little variation between the groups, reproducibility issues of differentiated U937 macrophages were evidenced by FCS batch-dependent significant (FDR (false discovery rate) < 0.05) differences in the formation of the chemokine CCL5, the cell growth regulator IGFBP2, and the cell migration and fibrinolysis regulator SERPINE1 (PAI1) and MMP1 (Figure 1A). In line, the amount of bioactive eicosanoids comprising the hydroxyeicosatetraenoic acids 11-, 12-, and 15-HETE, hydroxydocosahexaenoic acid 17-HDoHE, the prostaglandin PGJ2 and others were found to differ significantly (FDR < 0.05) depending on the FCS batch used for cell culture (Figure 1B). Table 1. Tested FCS batches stating Vender, Lot number, expiration date as well as letter used in this work. Figure 1. Heatmaps of selected proteins (A) and eicosanoids (B) determined in secretomes of control and LPS-treated U937 cells cultured with the indicated batch of FCS, A, B, C or D. Lines within a heatmap indicate a significant difference of the given molecule within at least two batches. Asterisks (\) indicate that LPS-treatment induced a significant increase. Venn diagrams of significantly up- and downregulated (C) proteins (S0 = 2, FDR = 0.01) and (D) eicosanoids comparing LPS activation with control samples for all four FCS batches (A–D). Inflammatory stimulation with LPS induced the secretion of a total of 67 proteins (FDR < 0.05, Supplementary Table S3), including tumor necrosis factors TNF and TNFSF15, chemokines such as CCL3, CXCL5, CXCL10, metalloproteinases including MMP1 and MMP10 and other promoters and mediators of inflammation (Figure 1A and Supplementary Table S3). Only 22 of those 67 proteins were found uniformly regulated independent of the FCS batch (Figure 1C), whereas other bioactive molecules such as IGFBP2 and TNFSF15 again showed FCS batch-dependent expression patterns (Figure 1C). Similarly, LPS treatment induced the formation of lipid mediators of inflammation such as 15-HETE, PGE2, PGJ2 and others (Figure 1B, Supplementary Table S4). The formation of eicosanoids varied rather strongly dependent on the FCS batch used, four out of seven LPS-induced eicosanoids showing significant batch-dependent alterations (FDR < 0.05, Figure 1D). #### 2.1. The Eicosanoid Content of FCS Varies in a Batch-Dependent Fashion* The induction of inflammatory activities of cells may be subject to modulation by a delicate balance of pro- and anti-inflammatory molecules. Thus we investigated whether the above-described batch effects may be caused by differences of the protein and eicosanoid content of the FCS batches used for the cell culture experiments. Remarkably, the protein profile comprising 289 identified proteins (FDR < 0.01) of the four different FCS batches was rather consistent (Figure 2A, Supplementary Table S5). Several significant abundance differences between batches were observed (Supplementary Table S5), and a principle component analysis showed fairly good clustering of the FCS samples according to batches (Figure 2B). The analysis of FCS eicosanoid contents revealed even stronger batch-dependent differences, comprising mainly COX and LOX-products (Figure 2C, Supplementary Table S6). Here, an unbiased PCA clustered the FCS batches with clear distances between batch clusters (Figure 2D), and demonstrated that batch dependent differences of eicosanoid content exceeded the differences of protein content. Figure 2. Radar plot for selected proteins (A) and fatty acids (B) identified in 4 different FCS batches without incubation with cells (baseline levels). Principal component analysis of protein (C) and eicosanoid (D) measurements of the same FCS batches, as indicated by different colors, demonstrates superior clustering in the case of eicosanoids. Asterisks mark significantly regulated molecules. #### 2.2. Cell Culture Subjects Fatty Acids to a High Turnover Growing cells require medium supply of fatty acids and fetal calf serum is rich in polyunsaturated fatty acids. In order to mediate biological effects as assumed for the FCS-contained fatty acids described above (Figure 2B), cells are supposed to take up fatty acids from the medium. In order to estimate to what extent cultured U937 cells may be able to take up fatty acids from the medium and release fatty acids back into the medium upon stimulation, we investigated the intracellular to extracellular exchange dynamics of the eicosanoid precursor molecule arachidonic acid (AA). For this purpose, cultured U937 cells were spiked with stable isotope labelled AA at a concentration double that of the endogenous AA (1.6 μM). Stable isotope labelled AA can be clearly distinguished from endogenous AA by mass spectrometry. As demonstrated in Figure 3, upon differentiation to macrophages, U937 cells apparently picked up more than 99% of the labelled and spiked AA within 72 h and less than 1% C13-AA remained detectable in the medium after that period. As expected, subsequent LPS treatment triggered a significant increase of the amount of C13-AA in the supernatant (Figure 3). This demonstrated that phospholipase activity was capable of releasing previously incorporated AA back into the medium. When supplementation with C13-AA was performed after PMA differentiation but before LPS treatment, the outcome was similar. Around 95% of the available AA was incorporated, but still a significant release of C13-AA was observed upon LPS treatment, clearly indicating a high turnover rate of AA. Concomitant measurement of endogenous C12-AA confirmed that AA was consumed substantially during cell culture and released back in the medium again in a smaller proportion upon LPS treatment. Figure 3. Experimental setup and results from AA spiking experiments. Medium was supplemented with 13C AA either before differentiation (left hand side) or before LPS treatment (right hand side). AA determination of cell supernatants by LC-MS/MS revealed AUC values as indicated. Medium levels at the beginning of cell culture are indicated by lines. Error bars are derived from three independent experiments. Con, untreated cells; act, LPS-treated cells. Note that AA concentration values strongly decrease upon cell cultivation but increase again upon LPS treatment. #### 2.3. Supplementation of 5-HETE and 15-HETE in the Nanomolar Concentration Range Induces the Formation of Peroxisomes in U937 Macrophages In order to demonstrate that the detected differences in eicosanoid content of FCS could originate from the observed batch effects, we performed an additional proteome profiling experiment with U937 cells at conditions only differing with regard to two selected eicosanoids, 5-HETE and 15-HETE. To this aim, we supplemented the FCS batch found to have the lowest levels of these two eicosanoids, here designated FCS-B (containing 5 nM 5-HETE and 12 nM 15-HETE), with the pure chemicals to levels close to those observed in case of FCS batch A (FCS-A, containing 42 nM 5-HETE and 49 nM 15-HETE, Figure 4A). Thus, U937 cells were grown and differentiated as before, using either FCS-A, supplemented FCS-A, or FCS-B, and subsequently subjected to proteome profiling of cytoplasmic proteins (Figure 4B). Indeed, spiking in of the two HETEs was associated with distinct proteome alterations (Figure 4B) including down-regulation of PKM and up-regulation of PEX 16, a peroxisomal membrane biogenesis protein [18]. To independently verify with a complementary method that this was a relevant observation, peroxisome formation was analyzed using immunofluorescence with an anti-PMP70 antibody. Nuclei and mitochondria were additionally stained to demonstrate uniform appearance of these organelles serving as background control. Indeed, treatment of U937 cells with increasing concentration of HETEs induced the formation of peroxisomes in an apparently concentration dependent manner (Figure 4C). Figure 4. (A) Eicosanoid levels of 5-HETE and 15-HETE for FCS-B (before spiking), FCS-A and FCS-B after spiking with 5-HETE and 15-HETE. (B) Volcano plot for cytoplasmic proteins obtained from U937 cells after PMA differentiation when cultured in either FCS-B or FCS-B supplemented HETEs. Bar plots exemplify the significantly regulated proteins PKM and PEX16. (C) Immunofluorescence detection of peroxisomes (red, PMP70 antibody), mitochondria (green, TOM20 antibody) and nuclei (blue, DAPI) shown for control and increasing concentrations of supplemented HETEs (addition of 1, 3, or 10 times of the spiked HETE mix). #### 3. Discussion The present data demonstrates that variations in the eicosanoid content of FCS may account for substantial batch effects with regard to functional readouts of a cell culture model reporting inflammatory mediators. This finding may be of great relevance for a large number of laboratories working with cell culture and using FCS, as FCS-contained eicosanoids have hardly been considered to have major implications for cell culture experiments and have thus, to the best of our knowledge, not yet been subjected to rigorous control. There are reasons, why relevant effects of eicosanoids contained in FCS were hardly expected. First, these molecules are generally considered to be short-lived and to act mainly in situ [19]. Second, eicosanoids were detected in FCS in the lower nanomolar concentration range, this is much less than the concentration range applied for functional assays in vitro, which is typically around 1 μM [20,21]. Furthermore, fatty acids including eicosanoids contained in serum are bound to albumin and only about 0.1% is actually free from associated molecules [22–24]. This free pool has a high turnover rate of about 2 min accounting for the redistribution of albumin-bound fatty acids in vivo to distant organs such as muscles or the liver. When investigating FCS batch effects, we initially expected proteins to represent the most plausible candidates contributing to reproducibility issues. Proteins as well as metabolites are strictly regulated in vivo to ensure homeostasis and consequently stable viability of the organism. While proteins may be rather stable in biological environments, metabolites such as fatty acids are much more vulnerable to chemical reactions such as oxidation, which may occur also during processing of FCS and are hard to control. Thus it was somewhat unexpected to see that FCS eicosanoid profiles were stable and clustered the FCS samples according to batches (Figure 2). This finding, supported by older and current literature reporting remarkable biological effects of eicosanoids [25–27], motivated us to focus on this class of molecules. Functional analyses were based on spiking experiments with the U937 cells. As a first step, the efficient and fast uptake of albumin bound arachidonic acid (AA) was verified in the present cell model system using stable isotope labelled AA. The subsequent release of labelled AA upon LPS stimulation of the cells strongly indicated the previous uptake and incorporation of AA into more complex lipids, from where AA was apparently released by the action of LPS-induced phospholipase A2 [28]. In order to test potential biological effects of eicosanoids on U937 cells, a decision was made in favor of commercially available eicosanoids, 5-HETE and 15-HETE, which were found to show remarkable concentration differences among the FCS batches. Hydroxyeicosatetraenoic acids (HETEs) are formed with AA by the action of lipoxygenases ALOX5 and ALOX15, expressed typically by epithelial cells as well as phagocytes such as neutrophils and macrophages [29,30]. Beside their effects on cell proliferation and differentiation, they are known activators of PPARγ [31]. Actually, peroxisome proliferator-activated receptors are known to induce the uptake and metabolism of fatty acids and to strongly modulate immune functions [32]. As fatty acid metabolism takes place in peroxisomes [33], the 5-HETE/15-HETE induced up-regulation of PEX16 (Figure 4), a peroxisome biogenesis protein indicative for peroxisome proliferation [18], indicated that this treatment caused an increased demand for these organelles. The concomitant down-regulation of PKM (Figure 4), a key enzyme for glycolysis [34], may suggest that HETE-treatment of U937 cells induced a metabolic shift increasing beta oxidation and attenuating glycolysis. This interpretation was independently supported by the concentration-dependent HETE-induced formation of peroxisomes (Figure 4) observed by immunofluorescence staining using a PMP70 antibody [35]. #### 4. Conclusions and Outlook The present data demonstrate that batch-dependent differences of eicosanoids contained in FCS may have a profound effect on cellular functions as observed with the U937 in vitro cell culture model for differentiation and inflammatory stimulation. Eicosanoids affect many relevant cellular events far beyond that, suggesting that they may represent the main contributors for reproducibility issues in cell culture. The establishment of a strict quality control regime controlling eicosanoid content in FCS may alleviate this challenging problem. #### 5. Materials and Methods #### 5.1. Cell Culture U937 cell line was cultured in RPMI medium (1X with L-Glutamine; Gibco, Thermo Fischer Scientific, Vienna, Austria) supplemented with 1% Penicillin/Streptomycin (Sigma-Aldrich, Austria) and 10% Fetal Calf Serum (FCS, Sigma-Aldrich, Vienna, Austria) in T25 polystyrene cell culture flasks for suspension cells (Sarstedt, Wiener Neudorf, Austria) at 37 ◦C and 5% CO2. Cells were counted with a MOXI Z Mini Automated Cell Counter (ORFLO Technologies, Ketchum, ID, USA) using Moxi Z Type M Cassettes (ORFLO Technologies, Ketchum, ID, USA) and the number of seeded cells for the experiments calculated based of these results. For all experiments the cells were used in passages 22–26. #### 5.2. Differentiation with Phorbol 12-Myristate 13-Acetate (PMA) and Inflammatory Activation with Lipopolysaccharides (LPS) All experiments were carried out in triplicates of LPS activation and control. For the proteomics and eicosadomics measurements 2 × 106 cells were seeded in T25 polystyrene cell culture flasks with cell growth surface for adherent cells (Sarstedt, Wiener Neudorf, Austria) with 5 mL fully supplemented media and 100 ng/mL PMA (Phorbol 12-myristate 13-acetate ≥ 99%, Sigma-Aldrich, Vienna, Austria) to induce differentiation. After 48 h incubation the medium was withdrawn and used for eicosanoid measurements. Three ml of new fully supplemented media was added either with 1 μg/mL LPS (Lipopolysaccharides from Escherichia coli 055:B5, γ-irradiated, BioXtra, Sigma-Aldrich, Vienna, Austria) or 1 μL PBS per 1 mL medium as control. After 24 h activation the medium was withdrawn again and used for eicosanoid measurements. The cells were gently washed twice with 5 mL phosphate buffered saline (PBS) and 3 mL new medium without FCS was added and incubated. After 4 h the supernatant was withdrawn and used for proteomics measurements. The cells were used for a subcellular fractionation as described before and cytoplasm and nuclear fraction were used for proteomics analysis [36]. #### 5.3. Test of Different FCS Batches Throughout the experiments different suppliers and batches of FCS were used. Additional details concerning the FCS batches are listed in Table 1. FCS batches A-C were heat inactivated at 56 ◦C for 30 min, batch D was already bought heat inactivated. Also different concentrations of HETEs and labelled arachidonic acid were supplemented and the respective controls treated with the same amount of LC-MS grade methanol (5 μL/3 mL medium). The experimental workflow of PMA differentiation and LPS activation was done for every condition similarly, only exchanging the FCS batch, supplier or eicosanoid. Additionally, for every condition 3 aliquots (3 × 3 mL) of the fully supplemented media were used for eicosanoid measurements to determine the default levels of eicosanoids present. #### 5.4. C13 Labelled Arachidonic Acid For the investigation of the uptake and release of PUFAs an experiment was carried out with the supplementation of 1,2,3,4,5-13C arachidonic acid (C13 AA, Cayman chemicals, Ann Arbor, Michigan, USA). Whenever C13 AA was added, a control experiment was supplemented with the same concentration of unlabeled arachidonic acid. For the first experiment the C13 AA was added at a concentration of 1.6 μM (used for all AA supplementations) to the fully supplemented medium during the 48 h PMA differentiation step. This concentration is around double that of the endogenous arachidonic acid, thus the supplementation tripled the concentration of biologically active arachidonic acid. Afterwards, the now adherent cells were washed three times with PBS and medium without supplemented AA was added together with or without LPS for 24 h. For the second experiment the cells were differentiated with PMA in standard medium, washed three times with PBS and medium supplemented with C13 AA was added together with and without LPS for 24 h. The eicosanoids were collected and measured in the supplemented medium without incubation (t0), after 48 h PMA differentiation and after 24 h LPS activation. The experimental setup is illustrated in Figure 3. #### 5.5. Proteomics of Supernatant (SN), Cytoplasm (CYT), and Nuclear Extract (NE) For the proteomics sample preparation, the s-trap system (Protifi, Huntington, NY) was employed following the manufacturers protocol with slight modifications. The precipitated proteins were dissolved in lysis buffer (8 M Urea, 0.05 M triethylammonium bicarbonate (TEAB) and 5% sodium dodecyl sulfate SDS) and diluted to obtain a protein concentration of about 1 μg/μL. Twenty μg of protein was used for each digestion. First, the sample was reduced with 20 μL dithiothreitol DTT (Sigma-Aldrich) at a final concentration of 32 mM for 10 min at 95 ◦C. Afterwards, 5 μL iodoacetamide IAA (Sigma-Aldrich) was added to a final concentration of 54 mM and incubated for 30 min at 30 ◦C in the dark. After adding 4.5 μL 12% ortho-phosphoric acid (Sigma-Aldrich) and 297 μl S- Trap buffer (90% Methanol (v/v) in H2O and 0.1 M TEAB) the sample was loaded onto the S-Trap Filter. The S-trap filters were centrifuged at 4000 xg for 1 min to pass through all the sample and trap the proteins onto the resin and afterwards washed four times with 150 μL S-Trap buffer. Twenty μg aliquots of Trypsin/Lys-C (MS grade; Promega Corporation, Madison, WI, USA) were dissolved in 400 μL 50 mM TEAB and 20 μg of this solution was added directly onto the resin of the filter (corresponding to 1 μg Trypsin/Lys-C per sample) and incubated for 1 h at 47 ◦C. After finishing the digestion, the peptides were eluted with 40 μL of 50 mM TEAB followed by 40 μL of 0.2% formic acid (FA) in H2O and 35 μL of 50% (v/v) acetonitrile (ACN) with 0.2% FA in H2O. The peptides were dried for about 2 h with vacuum centrifugation and stored at −20 ◦C until LC-MS/MS measurement. #### 5.6. HPLC-MS/MS for Proteomics For the HPLC-MS/MS analysis the peptides were resolved in 5 μL 30% formic acid and diluted with 40 μL of mobile phase A (97.9% H2O, 2% acetonitrile, 0.1% formic acid). One μL for the supernatant samples and 5 μL of cytoplasmic and nuclear samples were injected into the Dionex UltiMate 3000 RSLCnano liquid chromatography (LC) system coupled to the QExactive Orbitrap MS (all Thermo Fisher Scientific, Austria). Peptides were trapped on a C18 2 cm × 100 μm precolumn and LC separation was performed on a 50 cm × 75 μm Pepmap100 analytical column (both Thermo Fisher Scientific, Austria). Separation was achieved applying a 43 min gradient from 7% to 40% mobile phase B (79.9% acetonitrile, 20% H2O, 0.1% formic acid) for supernatant samples and 95 min gradients from 8% to 40% mobile phase B for cytoplasmic and nuclear samples, both at a flow rate of 300 nL/min, resulting in a total run time of 85 min and 135 min, respectively. Mass spectrometric settings were the same for all fractions. The resolution on the MS1 level was set to 70,000 (at m/z = 200) with a scan range from 400 to 1400 m/z. The top eight abundant peptide ions were chosen for fragmentation at 30% normalized collision energy and resulting fragments analyzed in the Orbitrap at a resolution of 17,500 (at m/z = 200). #### 5.7. Proteomics Data Analysis Raw data were subjected to the freely available software MaxQuant (version 1.6.0.1) [37] utilizing the Andromeda search engine, which returns label free quantification (LFQ) values for each identified protein as subsequently used for further data evaluation. For the MaxQuant search, a minimum of two peptide identifications, at least one of them being a unique peptide, was required for valid protein identification. Digestion mode was set to "Specific" choosing Trypsin/P. The peptide mass tolerance was set to 50 ppm for the first search and to 25 ppm for the main search. The false discovery rate (FDR) was set to 0.01 both on peptide and protein level. The database applied for the search was the human Uniprot database (version 03/2018, reviewed entries only) with 20,316 protein entries. Further settings for the search included carbamidomethylation as fixed modification and oxidation of methionine and acetylation of the protein C terminus as variable modifications. Each peptide was allowed to have a maximum of two missed cleavages and two modifications, "Match between runs" was enabled and the alignment window set to 10 min, with the match time window of 1 min. Statistical evaluation was performed with Perseus software (version 1.6.0.2) [38] using LFQ intensities of the MaxQuant result file. After filtering potential contaminants, the LFQ values were Log(2)-transformed. Technical duplicates were averaged. Only proteins detected in three of three biological replicates in either control and/or treatment groups were considered for data evaluation. Permutation-based FDR was set to 0.05 for t-tests and provided significant protein expression changes corrected for multi-parameters (S0 = 0.1). The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [39] with the dataset identifier PXD020617 and 10.6019/PXD020617. #### 5.8. Eicosanoid Sample Preparation Cell supernatants were spiked with 5 μL of internal standards (Supplementary Table S1) and centrifuged at 726 g for 5 min to remove cells and debris. Three ml of the supernatant was mixed with 12 mL of ice cold ethanol and left at −20 ◦C overnight to precipitate the contained proteins. The samples were centrifuged for 30 min with 4536 xg at 4 ◦C and the supernatant transferred into a new 15 mL Falcon tube. Ethanol was evaporated via vacuum centrifugation at 37 ◦C until the original sample volume was restored. Samples were loaded on conditioned 30 mg/mL StrataX solid phase extraction (SPE) columns (Phenomenex, Torrance, CA, USA). Columns were washed with 2 mL MS grade water and eicosanoids were eluted with 500 μL methanol (MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using N2 stream at room temperature and reconstituted in 150 μL reconstitution buffer (H2O/ACN/MeOH + 0.2% FA—65:31,5:3,5), containing a second set of internal eicosanoid standards at a concentration of 10–100 nM (Supplementary Table S1). #### 5.9. UHPLC-MS/MS for Eicosanoid Measurements Analytes were separated using a Thermo Scientific Vanquish (UHPLC) system and a Kinetex® C18-column (2.6 <sup>μ</sup>m C18 100 Å, LC Column 150 × 2.1 mm; Phenomenex®). Applying a 20 min gradient flow method, starting at 35% B steadily increasing to 90% B (1–10 min), going up to 99% B in 0.25 min. Flow rate was kept at 200 μL/min, 20 μL injection volume and column oven temperature was set to 40 ◦C. Eluent A contains H2O + 0.2% FA and eluent B ACN:MeOH (90:10) + 0.2% FA. Mass Spectrometric analysis was performed with a Q Exactive HF Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Austria), equipped with a HESI source for negative ionization. Mass spectra were recorded operating from 250 to 700 m/z at a resolution of 60,000 @ 200 m/z on MS1 level. The two most abundant precursor ions were selected for fragmentation (HCD 24 normalized collision energy), preferentially molecules from an inclusion list which contained 32 m/z values specific for eicosanoids (Supplementary Table S2). MS2 was operated at a resolution of 15,000 @ 200 m/z. For negative ionization, a spray voltage of 2.2 kV and a capillary temperature of 253 ◦C were applied, with the sheath gas set to 46 and the auxiliary gas to 10 arbitrary units. Generated raw files were analyzed manually using Thermo Xcalibur 4.1.31.9 (Qual browser), comparing reference spectra from the Lipid Maps depository library from July 2018 [40]. For peak integration and quantitative data analysis the software TraceFinderTM (version 4.1-Thermo Scientific, Austria) was used. For the quantification of arachidonic acid (Figure 3), a calibration curve was generated (Supplementary Figure S4). #### 5.10. Immunofluorescence For fluorescence microscopy 8 × 104 cells in 400 <sup>μ</sup>L were seeded in a <sup>μ</sup>-Slide 8 well (Ibitreat coating, ibidi GmbH Martinsried, Germany). Differentiation of the cells was induced with 100 ng/mL PMA for 48 h. Afterwards the cell supernatant was exchanged with fully supplemented medium without PMA for an additional 24 h. Sample preparation was performed as previously described with minor modifications [41]. Cells were fixed with pre-warmed formaldehyde (3.7%) for 15 min and permeabilized with Triton-X 100 (0.2%) for 10 min. Blocking was performed with Donkey serum (2% in PBS-A) for 1 h, room temperature (RT). Primary antibodies were incubated 2h at RT at dilution 1:500. After washing, specie-specific fluorescent-labelled secondary antibodies were added and slides incubated in a dark humidified chamber for 1.5 h. For our study, Anti PMP70 Antibody (Rabbit polyclonal, PA1-650) and Anti TOM20 (F-10, Mouse Monoclonal Sc-17764), Alexa Fluor 488 Donkey Anti Mouse (A21202\_LOT2090565) and Alexa Fluor 568 Donkey Anti-Rabbit (A10042\_LOT2136776) were used. The slides were washed and postfixed with 3.7% formaldehyde (10 min, RT); at the end of the post-fixation, 100 mM glycine was used to mask reactive sites and slides were mounted and sealed with Roti-Mount FluoCare (Roth, Graz, Austria) with DAPI. SIM Images were acquired with a Confocal LSM Zeiss 710 equipped with ELYRA PS. 1 system. Structured Illumination Microscopy (SIM) images were obtained with (Plan Apochromat 63X/1.4 oil objective) grid 5 rotation. For the quantification of fluorescence intensities (Figure 4B), 30 optical fields/region of interest (ROI) were quantified for every experimental condition from at least 3 independent experiments. #### 5.11. Differentiation Status by Flow Cytometry In order to confirm the differentiation status obtained via PMA treatment the cells were tested for the differentiation marker CD11b (ITGAM) using FACS analysis. Therefore, U937 cells were treated with 100 ng/mL PMA for 48 h using 2 × 105 cells per well in 6-well plates. After the incubation time, the cells were washed three times with PBS and put on ice. The differentiation status was assessed by labelling with an anti-CD11b antibody (APC clone D12, BD Bioscience) and subsequent evaluation of the CD11b+ population. Three biological replicates were analyzed per condition on an FACS Canto II cytometer (BD Bioscience). Supplementary Materials: The following are available online at https://www.mdpi.com/2218-273 X/11/1/113/s1, Figure S1: FACS analysis of PMA-treated U937 cells, Table S1: Eicosanoid Standards, Table S2: Inclusion list of eicosanoids as used for the MS/MS analysis, Table S3: Results from proteomic analyses of U937 cells, Table S4: Results from eicosanoid analyses of U937 cells, Table S5: Results from proteomic analyses of FCS batches, Table S6: Results from eicosanoid analyses of FCS batches. Author Contributions: L.N. performed research, analyzed and interpreted data, B.N. performed research, analyzed and interpreted data, J.B. performed research and analyzed data, L.J. performed research and analyzed data, A.B. interpreted data and wrote manuscript, G.D.F. performed research and analyzed data, C.G. conceptualized the project, interpreted data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [39] with the dataset identifier PXD020617 and 10.6019/PXD020617. Acknowledgments: We acknowledge support by the Mass Spectrometry Center and the Core Facility Multimodal Imaging of the Faculty of Chemistry, University of Vienna, as well as support by the Joint Metabolome Facility of the University of Vienna and Medical University of Vienna, all members of the Vienna Life Science Instruments (VLSI). Conflicts of Interest: The authors declare no conflict of interest. #### Abbreviations #### References ## Article Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels Benjamin Neuditschko 1,2,†, Marlene Leibetseder 1,†, Julia Brunmair 1, Gerhard Hagn 1, Lukas Skos 1, Marlene C. Gerner 3, Samuel M. Meier-Menches 1,2,4, Iveta Yotova <sup>5</sup> and Christopher Gerner 1,4,\* Abstract: Endometriosis is a benign disease affecting one in ten women of reproductive age worldwide. Although the pain level is not correlated to the extent of the disease, it is still one of the cardinal symptoms strongly affecting the patients' quality of life. Yet, a molecular mechanism of this pathology, including the formation of pain, remains to be defined. Recent studies have indicated a close interaction between newly generated nerve cells and macrophages, leading to neurogenic inflammation in the pelvic area. In this context, the responsiveness of an endometriotic cell culture model was characterized upon inflammatory stimulation by employing a multi-omics approach, including proteomics, metabolomics and eicosanoid analysis. Differential proteomic profiling of the 12-Z endometriotic cell line treated with TNFα and IL1β unexpectedly showed that the inflammatory stimulation was able to induce a protein signature associated with neuroangiogenesis, specifically including neuropilins (NRP1/2). Untargeted metabolomic profiling in the same setup further revealed that the endometriotic cells were capable of the autonomous production of 7,8-dihydrobiopterin (BH2), 7,8-dihydroneopterin, normetanephrine and epinephrine. These metabolites are related to the development of neuropathic pain and the former three were found up-regulated upon inflammatory stimulation. Additionally, 12-Z cells were found to secrete the mono-oxygenated oxylipin 16-HETE, a known inhibitor of neutrophil aggregation and adhesion. Thus, inflammatory stimulation of endometriotic 12-Z cells led to specific protein and metabolite expression changes suggesting a direct involvement of these epithelial-like cells in endometriosis pain development. Keywords: endometriosis; inflammation; metabolomics; multi-omics; proteomics #### 1. Introduction Endometriosis is a chronic inflammatory disease describing the abnormal growth of uterine tissue outside of the uterine cavity in the pelvic area [1]. Endometriotic cells are characterized by invasive phenotypes. They successfully attach to pelvic organs and cause pelvic inflammation [2,3]. Studies estimate that the disease is affecting about 1 in 10 women worldwide. The clinical symptoms include dysmenorrhea, dyspareunia, chronic pelvic pain and infertility. To date, there exists no curative treatment [1]. As one of Citation: Neuditschko, B.; Leibetseder, M.; Brunmair, J.; Hagn, G.; Skos, L.; Gerner, M.C.; Meier-Menches, S.M.; Yotova, I.; Gerner, C. Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels. Biomolecules 2021, 11, 1230. https:// doi.org/10.3390/biom11081230 Academic Editors: Peter Roman Jungblut and Vladimir N. Uversky Received: 20 July 2021 Accepted: 13 August 2021 Published: 17 August 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). the cardinal symptoms, pain strongly affects the patients' quality of life and can only be treated symptomatically so far. A large fraction of the published studies focused on phenotypic investigations and the molecular mechanisms, especially those associated with pain development, remain to be fully elucidated [4]. According to the rAFS/ASRM system, the extent of the disease does not correlate with the pain level experienced by individual patients [5]. The severity of the pain sensation seems to be connected to a mixture of neuropathy, neurogenic inflammation, nociception and hyperalgesia [6]. It is known that the lesion's surrounding nerves are infiltrated and compressed by endometriotic cells [7]. Moreover, there is evidence that nascent nerve cells can be attracted by the endometriotic lesion. Their inflammatory activated state may also directly cause and transmit pain stimuli to the central nervous system [8]. Some nerve cells may even release proinflammatory factors, which ultimately lead to neurogenic inflammation and increased local vascular permeability, enhancing migration of ectopic endometrial cells [9–11]. Furthermore, it is well accepted that endometriosis-associated pain is directly linked to dorsal root ganglion neurons [12]. Immunological dysregulation seems to represent a main pathogenesis driver in endometriosis [13]. Normally, cell-mediated immune responses contribute to the elimination of immune invaders and clearance of ectopic endometrial tissue. In endometriosis, the clearance of endometrial tissue in the peritoneal cavity is abolished due to an impaired immune response at the site of implantation [14,15]. Deregulated T-cell immunity and a suppressed activity of macrophages and NK cells were found to contribute to this process [16]. The activation of an inflammatory response in the peritoneum of women with the disease leads to local production of cytokines, chemokines and growth factors that enhance the growth of the ectopic endometrial tissue by inhibiting normal apoptotic processes and promoting local angiogenesis and neurogenesis [17]. The macrophage-nervous axis in endometriosis is commonly accepted to be the main cause for disease-associated pain [18]. Indeed, nerve infiltration is positively correlated with high density of tissue-resident macrophages in the lesion [19]. Alternatively, these nerve fibres are attracted by the action of semaphorins. As semaphorins normally regulate axon migration, axonal growth and guidance, altered semaphorin secretion may lead to aberrant nervous innervation in endometriosis [18]. Infiltrating the endometriotic lesions, they secrete neuroangiogenic factors and create a neovasculative environment [20]. Herein, especially VEGF secretion plays an important role in axonal outgrowth functioning as a neurotrophic factor [21,22]. Once neuroangiogenesis and infiltration in nerve fibres was initiated by aberrant inflammatory signaling, the endometriotic lesion may be create its own altered microenvironment [23]. Macrophages secrete tumour necrosis factor α (TNFα) and interleukin-1β (IL1β) that contribute to disease progression [24,25]. These inflammatory cytokines can mediate neurogenic inflammation and secretion of further neuroangiogenic factors [26,27]. TNFα signaling increases the transient receptor potential vanilloid 1 (TRPV1) nociception in the dorsal root neurons which contributes to hyperalgesia and neuropathic pain sensation [28,29]. It was also found to sensitize sensory nerves to a constant induction of the action potential via TRPV1 in patients, mainly through overexpression of voltage-gated sodium channels [27]. TRPV1 expression is also increased in uterosacral ligaments in endometriosis patients [30] and it has been shown that pain is often driven by dorsal root ganglion neurons, in association with TRPV1 [12]. Neurogenesis seems to be at least partially responsible for neuropathic pain experiences [31,32]. Therefore, the molecular mechanisms of neurogenesis in endometriosis need to be comprehensively characterized to contribute towards novel therapy options in endometriosis pain management. During the last years proteomics and metabolomics analysis were applied to uncover markers for early detection of endometriosis and to understand the molecular changes associated with its pathophysiology [33–35]. However, the use of these omics techniques is still sparse in endometriosis and multi-omics profiling was not yet applied, to the best of our knowledge. Combining proteomic with metabolomic profiling is especially attractive, since it may support a functional interpretation of the involved pathways. In addition, signaling lipids are key players during inflamation and act in a concerted fashion with proteins [36,37]. Thus, we have applied an in-depth proteome, metabolome and eicosanoid profiling of the endometriotic epithelial cell line 12-Z to investigate and characterize their responsiveness towards inflammatory signals. The cell line was isolated from a patient with peritoneal endometriosis and immortalized by Starzinski-Powitz [38]. 12-Z was characterized as epithelial-like (cytokeratin-positive/E-cadherin negative) and using a matrigel assay it was shown that the cell line was highly invasive [38]. To simulate the inflammatory macrophage signaling, the cells were treated with the cytokines TNFα or IL1β, which are typically upregulated in endometriotic lesions of patients [39]. We provide proteomic and metabolic evidence that the endometriotic 12-Z cells may independently express key mediators of neuroangiogenesis and neuropathic pain. #### 2. Material & Methods #### 2.1. Cell Culture The 12-Z cell line was a generous gift of Dr. Anna Starzinski-Powitz (Goethe-Universität Frankfurt) [38]. Human epithelial endometriotic cell line 12-Z was cultivated in DMEM-F12 phenolredfree (Gibco, Thermo Fisher Scientific, Vienna, Austria) with 10% (v/v) heat inactivated fetal calf serum (FCS, SigmaAldrich, Vienna, Austria) and 1% (v/v) penicillin and streptomycin (Sigma-Aldrich, Vienna, Austria). Cultivation was done in humidified incubators at 37 ◦C and 5% CO2. The 12-Z cell line was grown in T75 polystyrene cell culture flasks with cell growth surface for adherent cells (Sarstedt, Wiener Neudorf, Austria). Cells were sub-cultured every 3–4 days at a 1:3 ratio. Cells were cultivated until they reached a concentration of 80% confluency. Cell counting was performed using a MOXI Z mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA) using Moxi Z Type M cassettes (ORFLO Technologies, Ketchum, ID, USA). Cells were routinely checked for mycoplasma contamination using MycoAlert™ mycoplasma detection kit (Szabo-Scanidc, Vienna, Austria). Cells were seeded on a 6-well plate with cell growth surface for adherent cells (Sarstedt, Wiener Neudorf, Austria) at a density of 300.000 cells/well in 3 mL growth medium. Inflammatory stimulation was applied for 48 h at a concentration of 10 ng mL−<sup>1</sup> with either TNFα or IL1β (both Sigma-Aldrich). #### 2.2. Proteomics After the indicated treatment, the cells were washed twice with PBS and fractionation was performed as previously described [40]. Isotonic lysis buffer (1 mL of 10mM HEPES/NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton × 100, protease and phosphatase inhibitor cocktail (Sigma-Aldrich)) was added to the cells, which were scraped using a cell scraper. Cell lysis was achieved using mechanical shear stress employing a syringe and a needle. After centrifugation at 2270× g for 5 min supernatant containing cytoplasmic proteins was precipitated in 4 mL of cold EtOH overnight. Protein sample preparation: The ethanolic protein suspension was centrifuged at 4536g for 30 min (4 ◦C). The supernatant was discarded, while the protein pellet was dried and resuspended in lysis buffer (8 M urea, 50 mM TEAB, 5% SDS). Protein concentration was determined using the bicinchoninic acid assay. An aliquot of the samples containing 20 μg protein was digested using a modified Protifi protocol [41]. In short, the samples were diluted to a concentration of 1 μg μL<sup>−</sup>1. The diluted sample (20 μg in 20 μL) were pipetted into a 1.5 mL Eppendorf microcentrifuge tube and 20 μL of DTT (64 mM) was added. The samples were heated for 10 min at 95 ◦C under constant shaking (300 rpm). The samples were cooled to room temperature, treated with iodacetamide (5 μL of 486 mM solution, and incubated in the dark for 30 min at 30 ◦C and 300 rpm. Afterwards, phosphoric acid (4.5 μL of 12%) was added, resulting in 1.2% final concentration of phosphoric acid. S-Trap buffer (297 μL, 90% MeOH (v/v), 0.1 M TEAB) was added to the solution. The sample was loaded on the Protifi S-Trap column and washed 4 times with S-trap buffer (150 μL). Trypsin/LysC (MS grade; Promega Corporation, Madison, WI, USA) was added in a 1:40 ratio (0.5 μg for 20 μg protein). After digestion for 2 h at 37 ◦C peptides were eluted, dried using a SpeedVac and stored until further analysis. Data acquisition: Dried peptides were reconstituted in 5 μL 30% formic acid, containing four synthetic peptides, for quality control [42]. The samples were further diluted with 40 μL mobile phase A (97.9% H2O, 2% acetonitrile, 0.1% formic acid). Peptides were analyzed with a Dionex UltiMate 3000 Nano LC system coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Vienna, Austria) using a previously published method [40]. In short, peptides were separated on a 50 cm × 75 μm PepMap100 analytical column (Thermo Fisher Scientific), at a flow rate of 300 nL min−1. Gradient elution of the peptides was achieved by increasing the mobile phase B (79.9% acetonitrile, 20% H2O, 0.1% formic acid) from 8% to 40%, with a total chromatographic run time of 135 min including washing and equilibration. Mass spectrometric resolution on the MS1 level was set to 70,000 (at m/z 200) with a scan range from m/z 400–1400. The 12 most abundant peptide ions were selected for fragmentation (Top12) at 30% normalized collision energy and analyzed in the Orbitrap at a resolution of 17,500 (at m/z 200). Data analysis: Data was analyzedanalyzed in settings as previously described [42]. Briefly, raw data was submitted to the freely available software MaxQuant (version 1.6.6.0) [43] utilizing the Andromeda search engine. A minimum of two peptide identifications, at least one of them being a unique peptide, was required for valid protein identification. The false discovery rate (FDR) was set to 0.01 on both peptide and protein level. Uniprot database (Human, version 03/2018, reviewed entries only, 20,316 protein entries) was used to generate the fasta file used for the search. For statistical data evaluation MaxQuant companion software Perseus (version 1.6.1.0) was used. Reverse sequences and potential contaminants as well as proteins identified only by site were removed. Label-free quantification (LFQ) values were converted to Log2(x) and technical replicates averaged. Proteins were filtered for valid values, keeping only proteins that were identified in at least three measurements of one sample group. Evaluation of regulatory events between different samples groups was achieved by two-sided t-tests using a FDR < 0.05 calculated by permutation-based test [44,45]. Significantly regulated proteins were further analyzed using the Cytoscape [46] plugin ClueGo [47] with default settings. Gene ontology for Biological Processes (GOBP) was used as search space with medium network specificity. Statistical options were set to two-sided hypergeometriy test with Bonferroni step down p-value correction. String [48] analysis was further used to display protein network connections of regulated proteins. STRING protein query of species homo sapiens was used with a confidence (score) cut-off of 0.4 and with 0 additional interactors allowed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [49] partner repository with the dataset identifier PXD022354 and 10.6019/PXD022354. #### 2.3. Eicosanoid Analysis Eicosanoid sample collection and preparation: Supernatants from cell culture experiments or medium (3 mL) was added to cold ethanol (12 mL, EtOH, abs. 99%, −20 ◦C; AustroAlco) containing an internal standard mixture of 12S-HETE-d8, 15S-Hete-d8, 5-Oxo-ETE-d7, 11.12-DiHETrE-d11, PGE-d4 and 20-HETE-d6 (each 100 nM; Cayman Europe, Tallinn, Estonia). The samples were stored over-night at −20 ◦C. After centrifugation (30 min, 5000 rpm, 4 ◦C), the supernatant was transferred to a new 15 mL Falcon™ tube and EtOH was evaporated via vacuum centrifugation (37 ◦C) until the original sample volume was accomplished. Samples were loaded on preconditioned StrataX solid phase extraction (SPE) columns (30 mg mL−1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. SPE columns were washed with MS grade water (3 mL) and elution of eicosanoids was performed with ice-cold methanol (500 μL, MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated under a gentle stream of nitrogen at room temperature and the samples were reconstituted in 150 μL reconstitution buffer (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5), including a second mixture of internal standards, including 5S-HETE-d8, 14.15-DiHETrE-d11 and 8-iso-PGF2a-d4 (10–100 nM; Cayman Europe, Tallinn, Estonia). Reconstituted samples were stored at +4 ◦C and measured subsequently via LC-MS/MS. Data acquisition: Separation of eicosanoids was performed on a Thermo Scientific™ Vanquish™ (UHPLC) system equipped with a Kinetex® C18-column (2.6 μm C18 100 Å, LC Column 150 × 2.1 mm; Phenomenex®). All samples were analyzed in technical duplicates. The injection volume was 20 μL and the flow rate was kept at 200 μL min<sup>−</sup>1. The UHPLC method included a gradient flow profile (mobile phase A: H2O + 0.2% FA, mobile phase B: ACN:MeOH (vol% 90:10) + 0.2% FA) starting at 35% B and increasing to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Afterwards solvent B was decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min. The column oven temperature was set to 40 ◦C. The UHPLC system was coupled to a Q Exactive™ HF Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Austria), equipped with a HESI source for negative ionization to perform the mass spectrometric analysis. The resolution on the MS1 level was set to 60,000 (at m/z 200) with a scan range from m/z 250–700. The two most abundant precursor ions were picked for fragmentation (HCD 24 normalized collision energy), preferentially from an inclusion list containing m/z 31 values specific for eicosanoids and their precursor molecules. Resulting fragments were analyzed on the MS2 level at a resolution of 15,000 (at m/z 200). Operating in negative ionization mode, a spray voltage of 2.2 kV and a capillary temperature of 253 ◦C were applied. Sheath gas was set to 46 and the auxiliary gas to 10 (arbitrary units). Data analysis: Data interpretation of raw files generated by the Q Exactive™ HF Quadrupole-Orbitrap™ mass spectrometer was performed manually using Thermo Xcalibur™ 4.1.31.9 (Qual browser). Spectra were compared with reference spectra from the Lipid Maps depository library from July 2018 [50]. Peaks were integrated using the TraceFinder™ software package (version 4.1—Thermo Scientific, Vienna, Austria). #### 2.4. Metabolomics Metabolomic sample collection and preparation: For metabolomics analysis, cells were seeded at 10<sup>6</sup> cells per T25 flask in complete medium (5 mL) and left to adhere over-night. They were then treated with IL1β or TNFα for 48 h similarly to the proteomic experiment. Thereafter, the medium was removed and centrifuged (1100 rpm, 2 min, 4 ◦C). An aliquot (200 μL) of each medium sample was precipitated in cold MeOH (100%, 800 μL) and stored at −80 ◦C. The methanolic solution contained dopamine-d4, melatonin-d4 (both Santa Cruz Biotechnology, Dallas, TX, USA) and N-acetyl-serotonin-d3 (Toronto Research Chemicals BIOZOL) as internal standards at concentrations of 120 pg μL−1. The cell samples were washed once with PBS (3 mL) and metabolites were extracted with cold MeOH (80%, containing stds, 1 mL). The 80% methanolic solution contained dopamine-d4, melatonin-d4 (both Santa Cruz Biotechnology, Dallas, TX, USA) and N-acetylserotonin-d3 (Toronto Research Chemicals BIOZOL) as internal standards at concentrations of 100 pg μL<sup>−</sup>1. Each flask was processed at a time and immediately snap-frozen in liquid nitrogen. Three replicates per condition were then thawed together and the cells were scraped, transferred into labelled Eppendorf tubes and were stored at −80 ◦C. Samples were dried and reconstituted in 120 μL of 1% methanol and 0.2% formic acid and 1 pg μL−<sup>1</sup> caffeine-(trimethyl-D9) (Sigma Aldrich) and transferred into HPLC vials equipped with a 200 μL V-shape glass insert (both Macherey-Nagel GmbH Co. KG) suitable for LC-MS/MS analysis. Caffeine-(trimethyl-d9) (1 pg μL<sup>−</sup>1) was used as an additional internal standard. Again, the experiment was carried out in biological triplicates. Data acquisition: Samples were separated on a reversed phase Kinetex XB-C18 column (2.6 μm, 150 × 2.1 mm, 100 Å, Phenomenex Inc., Torrance, CA, USA) using a Vanquish UHPLC System (Thermo Fisher Scientific). Mass spectrometric analysis was performed on a Q Exactive HF orbitrap (Thermo Fisher Scientific). Mobile phase A consisted of water with 0.2% formic acid, mobile phase B of methanol with 0.2% formic acid and the following gradient program was run: 1 to 5% B in 0.5 min, 5 to 40% B from 0.5–5 min, then 40 to 90% B from 5–8 min, followed by a wash phase at 90% B for 2.5 min and then an equilibration phase at 1% B for 2 min, yielding a total run-time of 12.5 min. Flow rate was 0.5 mL min<sup>−</sup>1, injection volume was 5 μL and the column temperature was set to 40 ◦C. The injection needle was washed in between runs with 10% methanol. All samples were analyzed in technical replicates. Samples were analyzed in positive, as well as in negative ionization mode. Scan range was from m/z 100–1000 and resolution was set to 60,000 (at m/z 200) for MS1 and 15,000 (at m/z 200) for MS2. The four most abundant ions of the full scan were selected for further fragmentation in the HCD collision cell applying 30 eV normalized collision energy. Dynamic exclusion was applied for 6 s. Instrument control was performed using Xcalibur 4.0 Qual browser (Thermo Fisher Scientific). Data analysis: Raw files generated by the Q Exactive HF were loaded into the Compound Discoverer Software 3.1 (Thermo Fisher Scientific). Compounds were identified in Compound Discoverer with a user workflow tree. A maximum retention time shift of 0.1 min was allowed for aligning features and using a maximal mass tolerance of 5 ppm. Metabolites were matched against mzcloud (Copyright © 2021–2020 HighChem LLC, Slovakia mzCloud is a trademark of HighChem LLC, Bratislava, Slovakia). Compounds with a match factor ≥80 were manually checked. This was performed with Xcalibur 4.0 Qual browser (ThermoFisher Scientific). For peak integration and calculation of peak areas, the Tracefinder Software 4.1 (ThermoFisher Scientific) was used. The generated batch table was exported and further processed with Microsoft Excel, GraphPad Prism (Version 6.07) and the Perseus software (Version 1.6.12). #### 2.5. Cell Cycle Analysis Flow cytometry was performed to determine the cell cycle distribution with and without inflammatory stimulation. Therefore, BD Cycletest™ Plus DNA Kit (BD Biosciences, Vienna, Austria) was used according to the manufacturer's protocol to prepare the cells and measured on a CytoFLEX Flow Cytometer (Beckman&Coulter, Vienna, Austria) in the PE-channel. Statistical significance was evaluated with a bidirectional student t-test and three biological replicates. #### 3. Results Macrophages can stimulate endometriotic cells by secreting cytokines such as TNFα and IL1β [24,25]. Here, an in-vitro model of endometriosis was investigated to evaluate the effects of such inflammatory stimuli on endometriotic cells by means of a multi-omics approach, including untargeted shotgun proteomics, metabolomics and eicosanoid analysis (Figure S1). The endometriotic 12-Z cell line was treated with TNFα or IL1β at 10 ng mL−<sup>1</sup> for 48 h (Figure 1A). Flow cytometry analysis showed a slight increase in the tetraploid G2/M phase, as well as S-Phase and a corresponding decrease of cells in G0/G1 phase (Figure S2). #### 3.1. Eicosanoid Analysis Reveals the Uptake of Eicosanoid Precursors by 12-Z Cells from the Growth Medium Eicosanoids from the supernatants of control and inflammatory stimulated 12-Z cells were enriched by a solid-phase extraction protocol and subsequently analyzed by mass spectrometry. A total of 49 eicosanoids and polyunsaturated fatty acids (PUFAs) were detected in the supernatants. The composition of the fully supplemented medium was additionally verified. The epithelial-like 12-Z cells efficiently depleted the growth medium of the eicosanoid precursors arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) irrespective of treatment condition (Figure S2). Strikingly, EPA was not detectable after culturing the cells for 48 h. In contrast, the mono-oxygenated hydroxyeicosatetraenoic acids 16-HETE and 18-HETE were not detected in the growth medium, but only in the presence of the cultured cells. Inflammatory stimulation had little impact on the differential expression of eicosanoids. Figure 1. (A) Schematic representation of endometriotic 12-Z cells treated with TNFα (red) or IL1β (blue). IL-12 and NF-κB signaling was induced in both treatments while neuroangiogenesis was much more pronounced for TNFα stimulation. (B) Venn-Diagram showing the number of significantly regulated proteins (FDR = 0.05, S0 = 0.1) for TNFα (red) and IL1β (blue) compared to the control state. Twenty-seven protein groups were significantly regulated in both treatments. (C) Heatmap highlighting proteins involved in NF-κB and Il-12 signaling and downstream targets. (D) Protein signature characteristic for the phenotype of intermediate monocytes upon treatment of epithelial-like 12-Z cells with TNFα (red) and not with IL1β (blue). Asterisks (\) show multi-parameter corrected significant regulations of protein abundance (FDR = 0.05, S0 = 0.1) compared to untreated controls (Con). #### 3.2. TNFα- and IL1β-Stimulated 12-Z Cells Show Enhanced Proliferation and Activation of IL-12 and NF-κB Signaling Pathways* Proteomic profiling was performed on the cytoplasmic (soluble) fraction and resulted in the identification of 3684 protein groups. Label-free quantification (LFQ) was used to compare inflammatory stimulated with untreated conditions. In LFQ proteomics, the unlabeled peptides are quantified on the MS1 level and the intensities were adjusted to the overall intensity of all quantified peptides. Multi-parameter corrected statistical analysis (FDR = 0.05, S0 = 0.1) revealed 437 and 35 significantly regulated proteins after treatment with TNFα or IL1β, respectively, while 27 protein groups were regulated in both treatments (Figure 1B). Thus, inflammatory stimulation of 12-Z cells by TNFα led to 10-fold higher number of significantly regulated proteins compared to stimulation by IL1β. The most prominent regulations confirmed a successful inflammatory stimulation of the cells by upregulating IL-12 and NF-κB signaling pathways (Figure 1A). Upon TNFα treatment the classical (NFKB1) and alternative (NFKB2, RELB) NF-κB pathways were significantly upregulated while IL1β triggered partly the alternative pathway [51]. Both treatments, however, significantly induced the expression of downstream IL-12 and NF-κB targets (i.e., SOD2, SERPINB2) as exemplified in the heatmap in Figure 1C. Intermediate monocytes are a subpopulation of monocytes characterized by antigen presentation and transendothelial migration [52]. In contrast to classical monocytes, intermediate monocytes feature lower CD14 levels, but increased antigen presentation (HLA), lysozyme, S100A8 and S100A10 as identified by transcriptomic profiling. We found a strikingly similar protein signature corresponding to this intermediate monocyte state for the TNFα-treated, but not the IL1β-treated 12-Z cells (Figure 1D). #### 3.3. TNFα Induces the Expression of Proteins Involved in Neuroangiogenesis in 12-Z Cells TNFα stimulation showed the upregulation of several proteins involved in neuroangiogenesis. For example, regulated protein groups revealed dorsal root ganglion morphogenesis, sensory neuron axon guidance, neuron projection extension/guidance, semaphorin signaling and positive regulation of sprouting angiogenesis (Figure 2A). ClueGo analysis of significantly regulated proteins further revealed a network corresponding to angiogenesis, including amongst others the VEGF pathway (Figure 2B). Furthermore, the neuropilin receptors NRP1/NRP2 and RPL10 were found to be significantly upregulated [53]. STRING network analysis revealed a strong interconnection of proteins associated with neuroangiogenesis, which were significantly upregulated upon treatment with TNFα, including angiogenesis promoters ICAM1, VCAM1 [54], and ITGA5 [55] (Figure 2C). Furthermore, downstream targets of vascular endothelial growth factor (VEGF) signaling have been found upregulated upon stimulation with TNFα (e.g., BCRA1, ITGAV). A significant upregulation of proteins involved in semaphorin signaling was observed (e.g., OPTN, EPHA4, DHRS3). While none of the described proteins were significantly upregulated by IL1β treatment, they showed a similar trend, which highlighted the differential responses of these endometriotic cells to distinct inflammatory stimulations. Figure 2. (A) Gene Ontology terms for biological processes (GOBP) associated with neurogenesis. (B) ClueGo network for angiogenesis. (C) STRING network analysis for proteins involved in neurogenesis and angiogenesis. Red indicates multiparameter corrected significant regulation while yellow-colored proteins show higher expression but are not significant. #### 3.4. Untargeted Metabolomics Reveals the Upregulation of 7,8-Dihydroneopterin, 7,8-Dihydrobiopterin (BH2) and Normetanephrine in 12-Z Cells The signature of upregulated proteins involved in neuroangiogenesis and neuropathic pain motivated the investigation of pain-associated signaling molecules on the level of metabolites. For this purpose, an untargeted metabolomics assay was carried out by collecting whole cell lysates and supernatants of control and inflammatory stimulated 12-Z cells. Additionally, the fully supplemented medium was analyzed using the same method to determine the composition of the metabolic background similarly to the eicosanoid analysis. The experiment included a database search based on MS<sup>2</sup> fragment spectra which resulted in the identification of 29,607 features, from which the software annotated 633 compounds with a match factor ≥80 (Table S3). After manual review, 63 metabolites were selected and their abundances were quantified on MS<sup>1</sup> level as area under the curve based on accurate masses and retention time (Table S2). Multi-parameter corrected statis- tical analysis (FDR = 0.05, S0 = 0.1) revealed 15 and 4 significantly regulated metabolites for TNFα and IL1β in the whole cell lysates, respectively (Figure 3A). An analogous analysis of the supernatants revealed 3 and 26 significantly regulated metabolites for TNFα and IL1β treatments, respectively (Figure 3B). Strikingly, 7,8-dihydroneopterin, 7,8-dihydrobiopterin (BH2), epinephrine (identity of the molecule verified with external standards) and normetanephrine (annotated based on MS2) were detected in endometriotic 12-Z cells (Figures 3 and 4). These metabolites were not detected in the fully supplemented cell culture medium (Figure S3), but only in whole cell extracts and supernatants only in the presence of the 12-Z cells (Figure 4A). The recorded fragmentation spectra matched well the reference spectra from mzcloud database and corroborated the identification of these molecules (Figure 4B). In fact, BH2 and normetanephrine were significantly upregulated in the cellular interior during inflammatory stimulation with TNFα or IL1β, while 7,8-dihydroneopterin was upregulated upon TNFα stimulation only (Figures 3 and 4). The induction of BH2, 7,8-dihydroneopterin and normetanephrine was more pronounced upon activation with TNFα compared to IL1β. Interestingly, the enzymes involved in the biosynthesis of these metabolites remained largely constant upon inflammatory stimulation (e.g., dihydrofolate reductase DHFR or catechol O-methyltransferase COMT), with the exception of sepiapterin reductase (SPR), which was down-regulated by TNFα treatment (Figure 4). Epinephrine and normetanephrine were unexpectedly detected in 12-Z cells, as they were not yet associated with these endometriotic epithelial-like cells. Figure 3. Volcano plots comparing metabolite profiles of control experiments with TNFα and IL1β treatments in whole cell lysates (A) and supernatants (B). X-axis displays the calculated difference of treatment-control an a log2-scale and y-axis show the -log p-value for each molecule. Metabolites above significance curves represent multi-parameter corrected significantly regulated metabolites (FDR = 0.05, S0 = 0.1). Figure 4. (A) The metabolic pathway for the synthesis of 7,8-dihydroneopterin and 7,8-dihydrobiopterin is depicted together with the MS2 spectra of the measured metabolites compared to the reference spectrum from the mzcloud database. Intensity values of the metabolite and protein levels (orange background) in control, as well as TNFα– and IL1β-treatment, are given below. (B) Biosynthetic pathway of epinephrine derivatives. Normetanephrine is obtained from norepinephrine by the catechol-O-methyl transferase (COMT). MS<sup>2</sup> spectra of normetanephrine compared to the reference spectrum from the mzcloud database. Intensity values of the metabolite and protein levels (orange background) in control, as well as TNFα– and IL1β-treatment, are given below. None of the metabolites were detected in the fully supplemented medium (Figure S3). Asterisks (\) show multi-parameter corrected significant regulations of metabolite intensities compared to untreated controls (FDR = 0.05, S0 = 0.1). The orange shadows distinguish the abundance changes of proteins from those of the metabolites. #### 4. Discussion* Although endometriosis is affecting the quality of life of millions of women worldwide, representing a clear unmet medical need, the underlying molecular mechanism of this disease remains largely unknown. As pain sensation is among the most prevalent symptoms, investigating molecular mechanisms responsible for the development of pain may be key to identify useful therapeutic approaches. The interplay among endometriotic cells, macrophages and nerve cells in the ectopic lesions of endometriosis is of special interest for the origin of pain. We performed a multi-omics analysis, including proteomics, metabolomics and eicosanoid analysis, of the epithelial-like 12-Z endometriotic cell line in order to characterize the responses of these cells to inflammatory stimulation and their potential involvement in the development of pain. The 12-Z endometriotic cells were previously characterized as a proliferating and invasive cell line [38]. We found that inflammatory stimulation with TNAα or IL1β did not greatly affect the cell cycle distribution compared to untreated cells. In accordance, the eicosanoid precursors AA, DHA and EPA were efficiently depleted from growth medium irrespective of the inflammatory stimulus and were probably incorporated in the membranes of 12-Z cells. The mono-oxygenated 16– and 18-HETE are cytochrome P450 metabolic products of AA and were released from 12-Z cells. Importantly, 16-HETE is typically generated by exposure of resting neutrophils to AA [56] and represents an endogenous inhibitor of neutrophil activation [57] and thus exhibits anti-inflammatory effects. In our setup, the extent of 16-HETE release was independent of treatment condition. The proteomic data suggested that the 12-Z endometriotic cells, when stimulated with TNFα, may mimic an intermediate monocytic phenotype, which is generally characterized by transendothelial migration [52], and is actively involved in forming and sustaining a neuroangiogenic microenvironment characteristic for endometriotic lesions. Endometriotic cells were previously shown to exhibit enhanced migratory properties upon exposure to proinflammatory factors [9–11]. Moreover, dysfunction in macrophage-mediated phagocytosis of endometrial cells that undergo retrograde transport to the peritoneal cavity is considered an important factor in the development of endometriosis. In fact, this mimicry phenotype of the 12-Z cells may contribute towards the dysregulated immune clearance observed in endometriosis [14,15]. Generally, the 12-Z cells seem more susceptible towards stimulation by TNFα compared to IL1β. Inflammatory stimulation led to the upregulation of proteins involved in neuronal interactions as well as dorsal root ganglion morphogenesis and axon guidance. It is known that the pathology of endometriosis features neuronal interactions and especially neurogenesis [22]. Furthermore, a previous study already showed differential expression of semaphorins and neuropilin receptors correlating to dysmenorrhea [58]. Semaphorins are a group of evolutionarily highly conserved surface or locally secreted nerve repellent factors that can regulate axon migration, axonal growth and guidance [59–61]. The potential role of semaphorin 3A and its receptor (NRP1) in the regulation of aberrant sympathetic innervation in peritoneal endometriosis have been previously described [58]. Neuropilin receptors are prominent neurogenesis promoters, which function as axon guidance signaling receptors, as well as angiogenesis activation [31]. Our study shows that stimulation of 12-Z cells with TNFα upregulates the levels of NRP1, NRP2, DPYSL3, OPTN, EPHA4 and DHRS3 proteins suggesting an active involvement of endometriotic epithelial cells in semaphorin signaling. Normally, the process of nervous generation is a conserved feature present during embryonic development [62]. Although proteins like RPL10 and NRP1/2 are mostly associated with embryonic developmental signaling, they have been found significantly overexpressed upon TNFα stimulation in 12-Z in this study and suggest an unrecognized functional plasticity of these cells, which may contribute towards an increased understanding of this pathology. We further combined the proteome profiling with untargeted metabolomic analysis, investigating whether metabolites of 12-Z cells may be able to contribute to neuronal interaction and signaling. Especially, the capability of the production of 7,8-dihydrobiopterin (BH2), 7,8-dihydroneopterin, epinephrine and normetanephrine by endometriotic epithelial cells was striking. The differential expression of these metabolites was not correlated with the corresponding enzymes in their biosynthetic pathways (Figure 4). 7,8-Dihydroneopterin is an accepted metabolic inflammation marker normally generated by macrophages and has been related to impaired phagocytosis in endometriosis patients [14,63]. Epinephrine is a neurotransmitter secreted by the adrenal medulla. It is required for the vagus-mediated modulation of the nociceptive threshold and acts as inflammatory mediator induced in hyperalgesia [64]. Norepinephrine, the epinephrine precursor, from sympathetic nerve fibers is known to bind the oestradiol ß2 receptor on macrophages, leading to activation of PKA signaling and thus stimulating TNFα mediated inflammation [27]. Norepinephrine is generally involved in inflammation as well as endometriosis pathology [18,65]. This mechanism was previously described as an interaction between nerve cells and macrophages. The deregulation of epinephrine and semaphoring/NRP1 signaling pathways in the nerve cells of endometriosis lesion has been shown to support macrophage polarization [66–68]. Our data, however, suggest that epithelial endometriotic cells might themselves be capable of producing these metabolites, subsequently leading to enhanced TNFα secretion by polarized macrophages. The present model proposes not only a potential influence of endometriosis-associated epithelial cells on macrophages but on nerve cells as well. The significant upregulation of neurogenesis-related proteins demonstrated that the 12-Z cells may be capable of independently modulating neuronal mechanisms. Here again, norepinephrine and its metabolites might play an important role in the activation of pain [69]. It has been shown that epinephrine activates unmyelinated afferents in lesioned nerves [70]. Since endometriotic lesions contain large amounts of unmyelinated nerve fibres [71], there might be a connection between epinephrine secretion and the induction of pain sensation. The detection of epinephrine and normetanephrine in cell lysates and supernatants of IL1β-activated endometriotic cells is unprecedented, since this metabolite is normally produced by adrenal glands. Thus, TNFα and IL1β activation might be important to perpetuate the disease by affecting proteome, metabolome and eicosanoid levels differentially. Finally, the conversion of tetrahydrobiopterin (BH4) into BH2 is involved in the biosynthesis of norepinephrine as the initial hydroxylation step from tyrosine [72]. BH4 application in vivo has been shown to cause heat hypersensitivity and increased pain sensation through TRPV1 [73]. TRPV1 receptor is overexpressed in ectopic endometriosis implants, as well as in dorsal root ganglia of rats with endometriosis [12,74]. #### 5. Conclusions In summary, the data presented in this work highlights the proteomic, metabolomic and eicosanoid alterations upon inflammatory stimulation of the endometriotic epitheliallike cell line 12-Z. Besides the expected activation of inflammatory signaling cascades upon cytokine stimulation, these cells displayed an unexpected protein signature related to neuroangiogenesis which clearly underlined their capability to support neurogenesis in the lesion. Putative novel mediators in endometriosis pathology and pain development were discovered on the protein, metabolite and eicosanoid levels. This study indicates that 12-Z endometriotic cells may mimic an intermediate monocytic phenotype and actively participate in the crosstalk of the macrophage-nervous network within the lesion on the protein and metabolite levels. Thus, inflammatory stimulation of endometriotic cells by TNFα and IL1β seem to play an important role in the perpetuation of the characteristic inflammatory phenotype. They further seem to create factors enhancing the pain sensation through neurogenic inflammation. The actual interaction of endometriotic cells with macrophages and nerve cells requires further investigation but the presented data provided experimental evidence that they might be capable of hijacking immune cell functions in order to support the development and growth of an endometriotic lesion outside the uterine cavity. Supplementary Materials: The following supplementary information is available online at https: //www.mdpi.com/article/10.3390/biom11081230/s1: Figure S1: Schematic experimental setup, Figure S2: Cell cycle analysis and selected eicosanoids, Figure S3: Extracted ion chromatograms of selected metabolites; Table S1: Results from proteomic analysis, Table S2: Results from metabolomics analysis. Author Contributions: Conceptualisation, B.N., M.L., I.Y. and C.G.; Investigation, B.N., M.L., J.B., G.H., L.S. and M.C.G.; Formal analysis, B.N., M.L., J.B., G.H. and M.C.G.; Resources, I.Y. and C.G.; Validation, S.M.M.-M., C.G.; Visualisation, B.N., M.L. and S.M.M.-M.; Writing—original draft, B.N., M.L.; Writing—review & editing, S.M.M.-M., I.Y., C.G. All authors have read and agreed to the published version of the manuscript. Funding: Open Access Funding by the University of Vienna. Data Availability Statement: The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [39] with the dataset identifier PXD022354 and 10.6019/PXD022354. Acknowledgments: The authors are grateful to the Core Facility of Mass Spectrometry at the Faculty of Chemistry, University of Vienna and the Joint Metabolome Facility, University of Vienna and Medical University of Vienna. Both facilities are members of the Vienna Life-Science Instruments (VLSI). Conflicts of Interest: The authors declare no conflict of interest. #### References ## Article LPS Tolerance Inhibits Cellular Respiration and Induces Global Changes in the Macrophage Secretome Joseph Gillen 1,†, Thunnicha Ondee 2,†,‡, Devikala Gurusamy 3, Jiraphorn Issara-Amphorn 2, Nathan P. Manes 1, Sung Hwan Yoon 1, Asada Leelahavanichkul 2,4 and Aleksandra Nita-Lazar 1,\* Abstract: Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species. Keywords: host-pathogen interactions; proteomics; secretome; macrophages ### 1. Introduction Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial response to bacterial infection and tissue damage [1]. During Tolllike receptor (TLR) stimulation, macrophages are activated and produce pro-inflammatory cytokines and chemokines to recruit other cells to the site of infection [1,2]. In sepsis, lipopolysaccharides (LPS), an outer membrane component of Gram-negative bacteria, are considered to be a major activator of macrophages, triggering an inflammatory response [3]. However, in response to a second or prolonged LPS stimulation, macrophages are initially activated but produce lower amounts of pro-inflammatory cytokines. This phenomenon is called "LPS tolerance" or "endotoxin tolerance" and has been known since the 1940s [4–6]. While the lower cytokine production during LPS tolerance prevents a severe "cytokine storm" response and lethal effects in the host, decreased cytokine levels might not be Citation: Gillen, J.; Ondee, T.; Gurusamy, D.; Issara-Amphorn, J.; Manes, N.P.; Yoon, S.H.; Leelahavanichkul, A.; Nita-Lazar, A. LPS Tolerance Inhibits Cellular Respiration and Induces Global Changes in the Macrophage Secretome. Biomolecules 2021, 11, 164. https://doi.org/10.3390/ biom11020164 Academic Editors: Peter Jungblut and Michelle Hill Received: 1 December 2020 Accepted: 23 January 2021 Published: 27 January 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). sufficient to maintain an effective defense against pathogens. Indeed, LPS tolerance has been reported to be associated with the immune suppression stage known as immune exhaustion [6]. A concept of innate immunity bearing a memory of past insults termed "trained immunity" encompasses endotoxin tolerance, and its exploration may result in discoveries of new immunotherapies [7]. The mechanisms inhibiting the LPS response and moving cells into a tolerant state have still not been completely elucidated [8]. Findings from several groups emphasize the roles in this process of epigenetic reprogramming [4], microRNA [9,10], alteration of gene expression patterns [11], sometimes by specific transcription factors such as hypoxiainducible factor 1-alpha (HIF-1α) [12], non-coding RNAs [13], and energy depletion [14]. The metabolic changes in LPS-challenged macrophages after treatment with LPS have been indicated by several recent studies, with varying experimental designs focusing on a specific protein [15], pathway [16], or general phenotype [17]. Regulation of cellular signaling leads to changes in multiple secreted proteins that are responsible for the immune response during TLR stimulation (e.g., interleukin (IL)-6 and tumor necrosis factor (TNF) α). These proteins act as autocrine, paracrine, or chemoattracting signaling molecules for communication with other immune cells [18]. We have recently demonstrated the role of secreted lipocalin 2 (Lcn2) in the reduction in macrophage cytokine release in LPS-tolerant cells [15], but a comprehensive secretome analysis of LPS tolerance has not been previously reported. Investigating the secretome during tolerance induction could provide directions for explaining the phenomenon of immune tolerance and exhaustion. In this study, we used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of either macrophages responding to LPS stimulation or macrophages in the LPS-tolerant state. Next, we used mass spectrometry-based proteomics to thoroughly investigate, for the first time, the changes in the extracellular proteome (secretome) following the induction of LPS tolerance. Furthermore, we investigated the secretome profile during the induction of LPS tolerance to identify possible regulators of cellular metabolism and the production of proteins. In our analysis, we confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and reactive oxygen species metabolism. #### 2. Materials and Methods #### 2.1. Cell Culture and Reagents RAW 264.7 mouse macrophage cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum(FBS), 1 × glutamine, and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, referred to as complete DMEM (cDMEM). For stable isotope labeling by amino acids in cell culture (SILAC), the cells were cultured in DMEM for SILAC purchased from Thermo Fisher Scientific (Waltham, MA, USA), supplemented with 10% FBS, 1 × glutamine, 20 mM HEPES buffer, and isotopically labeled lysine and arginine purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). The cells were cultured in the labeled media for five passages prior to analysis to allow for > 95% incorporation of the labeled amino acids. Lipopolysaccharide (LPS) from Salmonella minnesota R595 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). #### 2.2. Quantification of Secreted Cytokines Secreted TNF-α, IL-6, and IL-10 were quantified with ELISA kits (Thermo-Scientific, Rockford, IL, USA) following the manufacturer's protocols. #### 2.3. Extracellular Flux Analysis The energy metabolism profiles of macrophages can be used to estimate glycolysis and mitochondrial oxidative phosphorylation on the basis of the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR), which were measured using Seahorse XF Analyzers (Agilent, Santa Clara, CA, USA). RAW 264.7 cells in different experimental groups, namely untreated (NT/NT or Con), LPS-responsive (NT/LPS or LR), and LPStolerant (LPS/LPS or LT), were dispersed into monolayers for measurement. A RAW mitochondrial stress test and a glucose stress test were performed at 37 ◦C using the Seahorse XFe96 bioanalyzer (Agilent, Santa Clara, CA, USA). RAW 264.7 cells in various treatment groups were collected and washed in 1× PBS. Cells seeded at 4 × 105 cells per well of the Seahorse analysis plates were centrifuged at 400 rpm with acceleration and deceleration set to 1 for 5 min to achieve an even monolayer of cells for accurate measurement. OCR and ECAR for the mitochondrial stress test were measured in xeno-free (XF) media (containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) under basal conditions and in response to 2 μM oligomycin, 1.5 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP), and 0.5 μM rotenone and antimycin A (Sigma-Aldrich, St. Louis, MO, USA). For the glucose stress test, the cells were cultured in XF media (containing 2 mM L-glutamine), and the ECAR readout was obtained at basal conditions and in response to 10 mM glucose, 1 μM oligomycin, and 10 mM 2-deoxy-glucose (2-DG). #### 2.4. Collection of Secreted Proteins For the secretome analysis by quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS), we used the method we established earlier [19]. Briefly, prior to stimulation, 1 × 106 RAW 264.7 cells were seeded in a well of a 12-well plate and grown at 37 ◦C for 24 h. To decrease the amount of non-specific protein in the media prior to stimulation, the cDMEM was removed from the cell culture wells and replaced with cDMEM lacking FBS. We extensively evaluated cell death in this method and found it to be negligible [19]. To test the effect of multiple stimulations of the innate immune system, the RAW 264.7 cells were treated in one of three ways. The first group contained control cells that received no LPS (NT/NT or Con) and were grown in cDMEM labeled with Arg0 and Lys0. The second group received a single stimulation with 100 ng/mL LPS 6 h prior to the collection of the media (NT/LPS or LR) and were grown in cDMEM labeled with Arg+6 and Lys+4. The third group received two stimulations with 100 ng/mL LPS separated by 24 h, with the second stimulation being 6 h prior to the sample collection (LPS/LPS or LT), and were grown in cDMEM labeled with Arg+10 and Lys+8. After the stimulations, the media were collected and equal parts of the three groups (v/v, as in [19–21]) were combined into a single 1.5-mL tube. Any cellular debris or detached cells were separated from the media by filtration using a 0.22-μm polysaccharide filter, and then, the medium was centrifuged at 400× g for 5 min. Finally, the supernatant was transferred to a 1.5-mL tube and the proteins were concentrated in a vacuum centrifuge (SpeedVac, Thermo Fisher Scientific, Waltham, MA) to dryness. Overall, this method was repeated twice with two biological replicates each time to produce four biological replicates. #### 2.5. In-Gel Digestion of Secreted Proteins The dried proteins were resuspended in 2 × NuPAGE loading buffer, and then, the proteins were denatured by boiling for 10 min. The proteins were separated using a 10% Bis-Tris NuPAGE gel (Invitrogen, 8 × 8 cm) with 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and run with 200 V for 40 min to ensure that there were no significant visual differences in the band patterns between samples. The gel was fixed using 47.5% methanol and 5% glacial acetic acid for 30 min at room temperature and then washed three times with ddH2O. The fixed proteins were stained with PageBlue protein staining solution (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature and then destained with ddH2O overnight at 4 ◦C. Following destaining, the lanes were cut from the gel using razor blades, sectioned into five equal units to avoid processing excess gel in one sample, and cubed into approximately 1-mm3 pieces. The gel pieces from each section were collected into 1.5-mL microcentrifuge tubes and then processed according to a previously published protocol [22]. In brief, 500 μL of acetonitrile (ACN) was added to the gel pieces and the tubes were incubated at room temperature for 10 min before a brief centrifugation and removal of the supernatant. Next, 50 μL of 10 mM dithriothreitol (DTT, Sigma-Aldrich, St. Louis, MO, USA) in 100 mM ammonium bicarbonate (ABC) was added to the gel pieces and the tubes were incubated at 56 ◦C for 30 min followed by a second incubation with ACN. Next, 50 μL of 55 mM 2-chloroacetamide (CA, Sigma-Aldrich, St. Louis, MO, USA) in 100 mM of ABC was added to the gel pieces and the tubes were incubated at room temperature in the dark for 20 min followed by a third incubation with ACN. Then, 100 μL of 50% ACN, 50 mM ABC was added to the gel pieces and the tubes were incubated at room temperature with occasional vortexing, followed by a fourth incubation with ACN. The gel pieces were saturated with 13 ng/μL sequence-grade modified trypsin (Promega; Madison, WI, USA) in 10 mM ABC, 10% ACN and the tubes were incubated at 37 ◦C overnight. To extract the peptides, 100 μL of a 1%:25% mix of formic acid:acetonitrile was added to the gel pieces and the tubes were incubated for 15 min in a 37 ◦C shaker. The tubes were centrifuged briefly and the supernatant was collected in 1.5-mL tubes. At this point, the peptides from the gel sections were recombined to make one sample per lane and the peptides were concentrated in a vacuum centrifuge (SpeedVac, Thermo Fisher Scientific, Waltham, MA, USA). Lastly, the samples were mixed with formic acid and ACN to generate peptide samples with a final concentration of 0.1% formic acid, 2% ACN. #### 2.6. Mass Spectrometry The Thermo Orbitrap Q-Exactive HF (Thermo Fisher Scientific, Bremen, Germany) and the Thermo UltiMate 3000 systems (Thermo Fisher Scientific, Bremen, Germany) were used for LC-MS/MS experiments. Peptides were trapped on an Acclaim C18 PepMap 100 trap column (5 μm, 100 Å, 300 μm i.d. × 5 mm, Thermo Fisher Scientific, Pittsburgh, PA, USA) and separated on a PepMap RSLC C18 column (2 μm, 100 Å, 75 μm i.d. × 50 cm, Thermo Fisher Scientific, Pittsburgh, PA, USA) at 40 ◦C. Peptides were eluted with a linear gradient of 2.5% to 5% mobile phase B (0.1% formic acid in ACN) for 15 min and then 5% to 35% mobile phase B over 90 min. Gradient changes were followed at 105 min to 35% mobile phase B and then increased to 99% mobile phase B at 110 min. The gradient was changed back to 2.5% mobile phase B at 125 min to equilibrate for 20 minutes prior to the next injection. Eluted peptides were ionized in positive ion polarity at a 2.3-kV spraying voltage. MS1 full scans were recorded in the range of m/z 400 to 1600 with a resolution of 60,000 at 200 m/z using the Orbitrap mass analyzer. Automatic gain control was set at <sup>1</sup> × 106 with 40 ms of maximum injection time. The top 20 data-dependent acquisition mode was used to maximize the number of MS2 spectra from each cycle. Higher-energy collision-induced dissociation (HCD) was used to fragment selected precursor ions with a normalized collision energy of 27%. Each biological replicate was analyzed twice to create two technical replicates. The mass spectrometry-based proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021925 [23]. #### 2.7. Analysis of MS Results The RAW MS files were processed with MaxQuant software (version 1.6.5.0, Max Planck Institute, Munich, Germany) [24] and searched with the Andromeda search engine [25] against a mouse UniProt FASTA database (download date: 26.03.2019, 22,325 entries) supplemented with common contaminants and reverse sequences of all entries [26]. The Andromeda search engine parameters were: type = three labels—light (Arg0, Lys0), medium—(Arg6, Lys4), and heavy—(Arg10, Lys8); fixed modification = carbamidomethylation of cysteine; variable modifications = oxidation of methionine, acetylation of lysine, and acetylation of protein N-terminus; minimum peptide length = 7; and max missed cleavages = 2. The false-discovery rate was set to 0.01 at the peptide spectrum matches (PSM), peptide, and protein levels. The protein group abundance data were filtered to remove possible protein contaminants. In addition, at least 2 identified peptides were required for each protein (if only one peptide was identified, at least 12 valid abundance values were required). This resulted in an estimated protein group false discovery rate of 1.54%. If a SILAC triplet contained one or two missing values, they were imputed by randomly generating a value between 10% and 100% of the minimum protein intensity value (equally distributed; separately for each LC-MS dataset). The abundance ratios were log2-transformed, and mean values were calculated across the technical replicates. InfernoRDN software v1.1.7626.35996 (https://omics.pnl.gov/software/infernordn) [27] was used to perform t-tests and to calculate post-hoc q-values. #### 3. Results #### 3.1. LPS-Tolerant Macrophages Decrease Cellular Respiration LPS tolerance is the decreased response of immune cells following secondary or prolonged stimulation with LPS. This process is typified by the decreased secretion of cytokines (Figure 1A–C), but the causes of tolerance and the processes that maintain it are not completely elucidated. This decreased response, although useful as a mechanism preventing a lethal outcome, can have tragic consequences for patients as the decreased secretion of cytokines might often lead to an increase in secondary infections. To examine the causes and regulation of LPS tolerance in macrophages, we used purified LPS to stimulate RAW 264.7 cells as an in vitro model, a methodology successfully used previously for secretome analysis with conclusions on changes in innate immune pathways and cellular metabolism by us [19] and others (for example, [21,28]). We found that while a single LPS stimulation enhanced cytokine release (LPS-Responding (LR)), two sequential LPS stimulations over a 24-h period induced decreased cytokine levels following a 6-h incubation (LPS-Tolerant (LT)) (Figure 1A–C). These results established the conditions required to induce LPS tolerance in RAW cells. While the decrease in secretion could be due to many factors such as a lack of available amino acids to build proteins, inhibition of vesicle transport, or increased turnover of specific mRNAs, we hypothesized that LPS-tolerant cells would display changes in their metabolic functions. The glycolytic and mitochondrial functions of control (Con or NT), LR, and LT cells were determined by measuring the ECAR and OCR using the Seahorse XF Extracellular Flux Analyzer. Both functions were impaired in LT cells compared to Con or LR cells (Figure 2A–E). Hence, the lower macrophage cytokine production in LPS-tolerant cells compared with control cells might be associated with the low cell energy. #### 3.2. Variations in LPS Treatment Lead to Variations in the Secretome To examine the conditions that contributed to the decreased respiration of LT cells, we analyzed the media collected from cells in each condition. By using SILAC metabolic labeling to mark each of the conditions prior to mass spectrometric analysis, as we have done in an earlier analysis of the TLR ligand-induced secretomes [19], we could simultaneously process and quantify the relative amounts of the proteins secreted by the cells in each condition (Figure 3A). We have reliably identified and quantified 1189 proteins across all conditions. Using a t-test to compare the intensities of the protein signals identified in the LR or LT samples to the Con samples, we found that several proteins had a two-fold or higher change in relative quantity and a significant change (p-value ≤ 0.05) versus the control (Figure 3B,C). In total, we found 56 and 107 proteins with significantly different levels in LR and LT cellular media, respectively. Figure 1. Induction of lipopolysaccharide (LPS) tolerance inhibits cytokine production by RAW 264.7 cells. Inflammatory cytokines in the supernatants (A–C) (n = 4/time point) of macrophages treated either once (LPS-Responding (LR)) or twice with LPS stimulation (LPS-Tolerant (LT)) and untreated control samples (Con), measured using ELISA kits, show significantly inhibited secretion of tumor necrosis factor (TNF)-α (A), interleukin (IL)-6 (B), and IL-10 (C) from LT cells compared to LR cells. \, p-value < 0.05 vs. LR; #, p-value < 0.001 vs. Con; φ, p-value < 0.05 vs. Con. Figure 2.* LPS-tolerant RAW 264.7 cells have significantly decreased cellular metabolism compared to LPS-responding and unstimulated cells. The general pattern of the estimation of glycolysis and mitochondrial functions through extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively (A,C); the pattern of macrophages treated with LPS either once (LPS-Responding (LR, red)) or twice (LPS-Tolerant (LT, green)) and untreated control samples (Con, blue) (B,D) (combination from triplicate experiments for B,D), and the energy map calculated using the Seahorse XF Extracellular Flux Assay (E). \* = p-value < 0.001. Figure 3. LPS-Responding and LPS-Tolerant RAW 264.7 cells secrete a wide variety of proteins. Schematic of the timing for LPS treatments of RAW 264.7 cells to induce the LPS response (LR) or LPS tolerance (LT) (A). MS analysis of the secretome of RAW 264.7 (B). Plot of t-test results comparing individual protein intensities calculated by MaxQuant (version 1.6.5.0) in LPS-Responding (LR) (C) or LPS-Tolerant (LT) (D) versus untreated control samples (NT/NT). Protein intensities of eight replicates were averaged and missing values for intrasample results were replaced with a random value between 1/2× and 2× the average of the 10 lowest values. The dotted lines indicate significance (p-value < 0.05) and the dashed lines indicate a onefold difference from the control. We found that most proteins were found in both treatment groups but had different levels and directions of change compared to the control in both LR and LT conditions. To identify which changes in protein levels were specific to either LR or LT conditions, we plotted the p-values versus the control of each protein in the LR and LT datasets (Figure 4A). This plot identified four clear groups, identified as A through D. Group A proteins had a significant difference (p-value ≤ 0.05) in LR samples and included 33 proteins. Group B had a significant difference (p-value ≤ 0.05) in LT samples and included 84 proteins. Group C had a significant difference (p-value ≤ 0.05) in samples treated with either LR or LT samples and included 23 proteins. Group D had no significant difference following LPS treatment and included 608 proteins (Figure 4A). Figure 4. LPS-Tolerant and LPS-Responding RAW 264.7 cells have distinct secretomes. Plotting of p-values of LR vs. Con against p-values of LT vs. Con confirms that only 19.3% of proteins have significantly modified secretion in both conditions (A). Of the proteins with significantly modified secretion in only LR cells, over half of the proteins have increased secretion (B). Of the proteins with significantly modified secretion in only LT cells, over half of the proteins have increased secretion (C). A comparison of the enrichment of proteins secreted by the LR and LT cells with significantly modified secretion shows that the majority of these proteins were significantly decreased following treatment (D). In addition to the significant difference from the control, the individual protein results could be further sorted by whether the intensity increased or decreased in comparison to the control (Figure 4B,C; Supplementary Table S1). The proteins that presented increased intensity following LPS treatments were termed subgroup one, while those with decreased intensity were termed subgroup two. Of the group A proteins, 18 were significantly increased (group "LPS-Responding Up" (LRU)) and 15 were significantly decreased (group "LPS-Responding Down" (LRD)). Group B proteins, while more numerous than those in group A, still had a bias towards increasing intensities, with 54 proteins from the group "LPS-Tolerant UP" (LTU) against 30 proteins from the group "LPS-Tolerant Down" (LTD). Lastly, for group C, significance following LPS treatment could lead to three possible outcomes: increased in both conditions (one protein), decreased in both conditions (18 proteins), or a discordant result with increased in one condition but decreased in the other condition (four proteins). Amongst the proteins in the LRU group were several cytokines and chemokines (Supplementary Table S1), including C-C motif chemokine 4 (Ccl4), tumor necrosis factor (TNF), C-X-C motif chemokine 10 (Cxcl10), C-C motif chemokine 2 (Ccl2), and leukemia inhibitory factor (LIF), which all showed significant (p-value < 0.05) or highly significant (p-value < 0.001) increases in their average intensity when compared to either the Con or the LT treatment group (Figure 4B, Supplementary Table S1). These data provide a perfect quality control for our dataset because these cytokines and chemokines are essential for the inflammatory response and are expected to be elevated in response to LPS. The group LRD included three proteins (Beta-glucuronidase (Gusb), beta-hexosaminidase, subunit alpha (Hexb), and alpha-N-acetylglucosaminidase (Naglu)) that localize to the phagolysosome and are associated with the metabolism of carbohydrates [29]. These proteins, along with vinculin (Vin), Dipeptidyl peptidase 2 (Dpp7), and malate dehydrogenase mitochondrial (Mdh2), displayed between a 1.25- and 5.75-fold significant decrease in intensity with t-test p-values ranging from 0.007 to 0.0484 following the LPS treatment (Supplementary Table S1). In contrast to the LR groups, the LT groups both contained many proteins typically found in the cytoplasm or other regions of the cell in addition to some secreted proteins (Supplementary Table S1). LTU proteins included 54 proteins with increases ranging from 48- to 2.78-fold versus the control sample and included osteopontin (Spp1), neutrophil gelatinase-associated lipocalin (Lcn2), sequestosome-1 (Sqstm1), and TAR DNA-binding protein 43 (Tarbp) (Figure 4C). The t-test p-values of each protein versus the control ranged from 0.0491 to 0.0001 (Supplementary Table S1). In contrast to the LTU proteins, nearly half (7/19) of the LTD proteins were associated with extracellular space. The 30 proteins from the LTD group showed between a 22- and 1.6-fold significant (p-values between 0.04 and 0.00001) decrease in overall intensity versus the control cells and included the urokinase-type plasminogen activator (Plau), sodium/potassium-transporting ATPase subunit gamma (Fxyd2), lysozyme C-2 (Lyz2), and cystatin-C (Cst3) (Supplementary Table S1). The last and smallest group of proteins that showed significant differences versus the control depending on the treatment with LPS were group C proteins (LPS-Dependent (LD)). The inclusion of the second treatment group leads to three possible results: both increase (LDU), both decrease (LDD), or one increases and one decreases (mixed) (LDM). In our analysis, we found only one LDU protein, plasminogen activator inhibitor 1 (Serpine1), and four LDM proteins, Talin-1 (Tln), MARCKS-related protein (Marcksl1), cytosolic nonspecific dipeptidase (Cndp2), and eukaryotic initiation factor 4A-I (Eif4a1), with increases in at least one treatment group by 29- to 1.6-fold versus the control set (Supplementary Table S1). The last group of 18 proteins identified in our analysis were the proteins with significant decreases in intensity (between 2000- and 1.5-fold) versus the control in both treatment conditions (group LDD), such as gelsolin (Gsn), low-density lipoprotein receptorrelated protein 1 (Lrp1), macrophage colony-stimulating factor 1 receptor (Csf1r), and fibronectin (Fn1) (Supplementary Table S1). #### 3.3. Pathway Analysis of Critical Groups Because either increasing or decreasing secretion of a signaling protein could have profound effects on the condition of cells, we analyzed all proteins with significant changes using the Ingenuity Pathway Analysis (IPA) software suite (Qiagen) (Figure 5). This analysis allowed us to identify several patterns, including pathways or functions enriched in either both or only one dataset. The canonical process associated with LPS treatment is the inflammatory response. While both LR and LT groups had highly significant effect changes in the inflammatory response (both p-values < 0.01), the LR group showed a strong increase (z-score of 1.908) and the LT group had a smaller increase (z-score of 0.204) (visualized in Figure 6A, Supplementary Table S2). When we focus on the myeloid cell responses, the differences in the LR and LT groups become even more striking. While the "Immune Response of Myeloid Cells" is significantly affected in either condition (p-values of <0.001), the LR condition had an increased response (z-score 1.134) but the LT condition had a decreased response (z-score −0.348) (visualized in Figure 6B, Supplementary Table S2). By examining a heatmap of the proteins measured from each condition, it was found that while the LR group had several signaling molecules, including CXCL3, CXCL10, and TNF, the LT group had decreased recovery of these signaling molecules along with decreased secretion (compared to the untreated control) of the urokinase-type plasminogen activator (PLAU) (Figure 6B), a secreted enzyme that activates plasmin, a protein that is critical for the complement system [30]. These results confirm that either type of LPS treatment induces the inflammatory response, but the response after sequential LPS treatment is significantly reduced. Figure 5. LPS-Responding RAW 264.7 cells have secretomes strongly associated with the immune response in contrast to LPS-Tolerant RAW 264.7 cells. Comparison of the Ingenuity Pathway Analysis of the proteins with significantly changed secretion in either LR or LT cells shows that while LR cells secreted proteins that strongly relate to the immune response and chemotaxis, LT cells secreted proteins that strongly relate to metabolism and cellular survival. Prepared using the Ingenuity Pathway Analysis program suite from QIAGEN (Germantown, MD, USA). Figure 6. LPS-Responding RAW 264.7 cells' secretomes include cytokines and signaling proteins strongly related to the inflammatory response and cell motility, while LPS-Tolerant RAW 264.7 cells' secretomes include proteins strongly related to cell survival. Fold changes of the proteins associated by Ingenuity Pathway Analysis to Inflammatory Response (A); Immune Response of Myeloid Cells (B); Cell Movement by Macrophages (C); Cell Death of Immune Cells (D); Clearance of Cells (E), and Respiratory Burst of Myeloid Cells (F). Prepared using the Ingenuity Pathway Analysis program suite from QIAGEN (Germantown, MD, USA). Another biological function associated with all three sets and with the LPS response was cellular motility. Due to the variety of cells and mechanisms of movement, most analysis platforms include both general terms and specific pathways. In the LR group, "Cell Movement of Macrophages" was significantly increased (p-value < 0.001, z-score 2.829), and while the LT group had a highly significant increase (p-value < 0.001), the overall degree of migration was lower (z-score −0.290). In our comparison, both datasets were associated with migration and contained at least five significantly elevated or decreased proteins (Figure 6C). This suggests that both treatments lead to cellular migration, but the overall effect was much higher in the LPS-responsive group. While the processes of inflammation and movement are critical for the immune response, cell survival has been the hypothetical goal of LPS tolerance. In support of this hypothesis, our results found significant inhibition of "Cell Death of Immune Cells" in the LT group (p-value < 0.01, z-score −0.254) (Supplementary Table S2). In contrast, the LR group had a highly significant increase in the "Cell Death of Immune Cells" (p-value < 0.001, z-score 0.565) (Supplementary Table S2). The difference in the recovery of cell-survivalassociated proteins from the LT and LR groups suggests a connection between cell survival and LPS tolerance (Figure 6D). #### 3.4. Relationship between Repeated LPS Stimulation and Cellular Exhaustion We have hypothesized that cellular exhaustion is related to suppression of the LPS response in sequential LPS treatments. Two pathways that relate to exhaustion are metabolism and the production of reactive oxygen species. By filtering the IPA comparative analysis results only for processes related to metabolism or reactive oxygen species, we found distinct differences between the LR and LT groups (Figure 7). Overall, the LT group has a wide variety of affected processes, with both increased and decreased rates predicted. Figure 7. LPS-Tolerant RAW 264.7 cell secretomes include proteins related to initiation of protein metabolism. Comparison of the Ingenuity Pathway Analysis of proteins with significantly changed secretion in either LR or LT cells shows that while LR cells secreted proteins strongly relate to carbohydrate metabolism, LT cells secreted proteins that strongly relate to protein and reactive oxygen species metabolism. Prepared using the Ingenuity Pathway Analysis program suite from QIAGEN (Germantown, MD, USA). Metabolism can be further defined by the class of molecule targeted, such as protein, lipid, or carbohydrate. The two classes that exhibited the clearest differences between the LR and LT groups were the processes related to carbohydrate and protein metabolism. In carbohydrate metabolism, the overall effect is that the LPS response induced increased carbohydrate metabolism, including the binding, accumulation, and metabolism of polysaccharides (Figure 8B). In contrast to the carbohydrate results, an examination of the processes related to protein metabolism showed increased association between the LT group and protein metabolism. Overall, protein metabolism appears to lean towards the accumulation of new proteins, with the increased z-score of overall protein metabolism and protein synthesis coinciding with decreases in protein catabolism and proteolysis (Figure 8C). The last aspect of metabolism with distinct differences between the LR and LT groups is the pathways related to reactive oxygen species. Overall, the LT group results were linked to lower metabolism and synthesis of ROS compared to the LR group (Figure 8D, Supplementary Table S2). These results confirm the modifications in the cellular environment that occur during both the LPS response and LPS tolerance. Based on the strong association of metabolism and reactive oxygen species with the previously shown effects of LPS tolerance on cellular respiration, we concluded that the induction and maintenance of LPS tolerance is dependent on the rates of cellular respiration, and further studies of the modifiers of cellular respiration and metabolic rates could lead to greater understanding of the regulation of LPS tolerance. Figure 8. LPS-Tolerant RAW 264.7 cell secretomes include proteins related to global changes in cellular metabolism. z-scores of the metabolic pathway results generated by Ingenuity Pathway Analysis show abnormal metabolism and Respiratory Burst of Myeloid Cells in LR cells (A), increased carbohydrate metabolism by LR cells (B), increased protein metabolism along with decreased protein translation by LT cells (C), and increased maintenance of reactive oxygen species by both cells (D). #### 4. Discussion LPS tolerance is a cellular condition defined by a lack of a typical immune response to LPS stimulation, originally characterized by decreased levels of secreted cytokines such as TNF-α, IL-6, and IL-10 (Figure 1). We have shown that LPS-tolerant RAW 264.7 cells secrete a wide variety of proteins, including several not typically found in the secretome, defined as proteins released from the cells as described by Koppenol-Raab et al. [19] (Figures 3 and 4). Similarly, while LPS-responding cells have basal metabolic rates the same as or above unstimulated control cells, LPS-tolerant cells show a significant decrease in their glycolytic and aerobic respirations (Figure 2). #### 4.1. Most Evident Protein Level Changes in the Secretome Using MS analysis combined with SILAC labeling to allow for direct comparisons of the Con (NT), LR, and LT secretomes, we identified global changes in the secretome following the induction of either the LPS response or LPS tolerance (Figure 3). It is important to note that the experimental setup with serum-free media necessary to facilitate mass spectrometry-based proteomics may affect the cell response. We have established that the cells respond to TLR ligands for up to 24 h, with the secretion patterns of known inflammatory cytokines being the same as the cells in the complete media [19], but there is a probability that some elements of the response to the second LPS stimulation, although many controls are as predicted for the LPS-tolerant state, may be changed by this variable. A comparison of the LR and LT secretomes further confirmed the vast differences in the quantity and types of secreted proteins that had significantly enhanced secretion (Figure 4). Using pathway analysis of the secreted proteins, we found that the LR cell secretome is highly associated with the innate immune response (Figures 5 and 6). In contrast, the LT cell secretome is highly associated with cell survival and modulation of cellular metabolism (Figures 5–7). These modulations focus on many aspects of both protein metabolism and reactive oxygen species metabolism (Figure 8). #### 4.2. Potential Protein Regulators of LPS Tolerance The clear differences between LR and LT cells raise the question of which signaling molecules induce and maintain LPS tolerance following multiple stimulations with LPS. Possible inducers or regulators of LPS tolerance could be secreted proteins (previously shown by either the protein itself, a closely related protein, or a homolog), specifically enriched in the LPS-tolerant cells, that have been previously linked to two or three of the critical functions we identified above (cell survival, protein metabolism, and maintenance of ROS). An examination of the LT group proteins identified several proteins that fulfill many of these requirements (Table 1). Three proteins that were linked to all three of the critical functions were superoxide dismutase 2 (SOD2, just below the statistical significance threshold but important to mention), sequestosome 1 (SQSTM1), and osteopontin 1 (SPP1). In addition, three secreted proteins were specifically enriched and involved in cell survival along with redox. The last group of seven proteins have been shown to be secreted, were specifically enriched, and were involved in cell survival along with protein metabolism. One protein with a direct association with the metabolism of protein and reactive oxygen species is mitochondrial superoxide dismutase (SOD2), whose deficiency has been linked to inflammatory disorders [31]. SOD2 has been shown to be increased in the process of the macrophage protection from reactive oxygen species-induced cell death [32]. Interestingly, its upregulation was described together with the downregulation of PARP1, an enzyme adding ADP-ribose to many proteins, a modification which we have recently shown to be regulated by LPS in macrophages [33]. Since we have found many proteins involved in the inhibition of apoptosis and necrosis in LPS-tolerant cells, there may be crucial mechanisms affected by proteins within this group that can be targeted for tolerance induction. Table 1. Potential regulators of LPS tolerance. Proteins with enhanced secretion by LPS-Tolerant RAW 264.7 cells vs. control and LPS-Responding cells that have associations with cell survival along with associations with either protein metabolism, reactive oxygen species metabolism, or both. "Secreted" results include "Yes" for proteins shown previously to be secreted in mice, "Yes (related)" for proteins shown previously to be secreted in humans, or "Yes (exosome)" for proteins shown previously to be secreted in exosomes in humans. The second secreted protein that affects all three processes is sequestosome 1 (SQSTM1, or p62), a receptor for selective autophagy that is responsible for sequestering cytoplasmic components into an autophagosome [34] and which, by its role in regulation of autophagy, affects macrophage survival. Because of these roles in autophagy, its upregulation following LPS tolerance would be another indication of the switch to survival mode. Additionally, SQSTSM1/p62 has been proposed to act as an inflammatory signaling platform after activation by transforming growth factor beta-activated kinase 1 (TAK1) (one of the kinases essential in TLR4 signaling [35]), effectively disabling it as an autophagy receptor and inhibiting its own degradation [36]. The final secreted protein that affects all three processes is osteopontin 1 (SPP1), a secreted bone matrix glycoprotein protein that is essential for bone homeostasis and control of cell migration [37,38]. SPP1 has also been shown to be expressed by macrophages during tissue repair after myocardial infarction [39], indicating its function in the tissue homeostasis function of macrophages as opposed to the inflammatory function. The secreted proteins may also provide an autocrine signal to balance cytokine production, a main feature of LPS tolerance. For example, in LT, the IL-1 receptor antagonist (Table 1) might directly decrease cytokine production [40], while Lipocalin-2 counteracts LT through the induction of cytokine production [15]. Hence, the understanding of these proteins and complex feedback loops is fundamental to control LPS signaling and macrophage function. The specificity of the secreted proteins associated with LPS tolerance does raise the question of the role of the regulation of protein signaling in leading to stimulation typespecific protein secretion in the initiation and maintenance of LPS tolerance. The role of post-translational modifications, especially phosphorylation, was pointed out as a regulatory mechanism in LPS tolerance nearly thirty years ago [41] and linked to crosstalk with other signaling pathways, for example, Fc gamma receptors (FcGRs) [42]. On the other hand, pathways intuitively associated with the regulation of the immune response may not be required for the induction of endotoxin tolerance, as shown for type 1 interferon signaling [43]. In addition to TLR4 signaling, NLRP3 inflammasome has been shown to play an important role in the response to LPS and has recently been shown to be regulated by specific lipid mediators [44]. These results may open another avenue for exploration of the mechanisms of LPS tolerance and for explanation of changes in the secretome. Cellular metabolism has recently emerged as a regulator of macrophage phenotype in general [45]. Unbiased secreted protein profiling and system-level characterization of changes in innate immune signaling and cellular metabolism, pointing to regulation at the post-transcriptional level, emphasize the importance of global studies that reach beyond gene expression analysis. Our study reveals the value of proteomics approaches that can explain rapid functional changes necessary for effective immune function. In the clinic, macrophage LPS tolerance could be either beneficial or harmful to the host, depending on other factors. While well-controlled LPS tolerance reduces overwhelming cytokine production (cytokine storm) and attenuates sepsis severity [46], unhinged LPS tolerance immune exhaustion might be harmful [47]. Novel ways to inhibit cytokine secretion and controlled induction of LPS tolerance should therefore be considered as a future treatment of septic shock. Supplementary Materials: The following are available online at https://www.mdpi.com/2218-273 X/11/2/164/s1, Supplementary Table S1. Sheet 1: All of the proteins identified in all the analyses (protein identifiers: Column C). Sheet 2: Protein result filtering to identify proteins with two or more peptides and 12 or more valid values. Sheets 3–5: Imputation of missing values and conversion of data into Log2 Fold changes in protein intensity recorded. Sheet 6: Summary of protein quantification and p-values for all the proteins quantified. Supplementary Table S2. All of the biological processes and cellular functions examined in the LPS-Responding and LPS-Tolerant cells, with p-values and significance marked with asterisks (increasing significance is indicated with more asterisks). Author Contributions: J.G.: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Visualisation, Writing—Original draft, Writing—review&editing; T.O.: Investigation, Methodology, Writing—Original draft, Writing—review&editing; D.G.: Investigation, Methodology, Visualisation, Writing—Original draft, Writing—review&editing; J.I.-A.: Investigation, Methodology, Writing—Original draft, Writing—review&editing; N.P.M.: author (6) Formal Analysis, Methodology, Visualisation, Writing—review&editing; S.H.Y.: Investigation, Methodology, Writing—Original draft, Writing—review&editing; A.L.: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing—review&editing; A.N.-L.: Conceptualization, Funding acquisition, Methodology, Project Administration, Resources, Supervision, Writing—Original draft, Writing—review&editing. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Intramural Research Program of NIAID, NIH, Thailand Government Fund (RSA-6080023), the Thailand Research Fund (RES\_61\_202\_30\_022), the Ratchadaphiseksomphot Endowment Fund 2017 (76001-HR), the Second Century Fund (C2F), Chulalongkorn University (to T. O.), and the Program Management Unit for Human Resources and Institutional Development Research and Innovation—CU (Global Partnership B16F630071 and Flagship B05F630073). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The mass spectrometry-based proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021925. Acknowledgments: This research was supported in part by the Intramural Research Program of NIAID, NIH. Conflicts of Interest: The authors declare no conflict of interest. #### References ## Article Omics Technologies to Decipher Regulatory Networks in Granulocytic Cell Differentiation Svetlana Novikova †, Olga Tikhonova †, Leonid Kurbatov, Tatiana Farafonova, Igor Vakhrushev, Alexey Lupatov, Konstantin Yarygin and Victor Zgoda \* > Orekhovich Institute of Biomedical Chemistry, Pogodinskaya 10, 119121 Moscow, Russia; [email protected] (S.N.); [email protected] (O.T.); [email protected] (L.K.); [email protected] (T.F.); [email protected] (I.V.); [email protected] (A.L.); [email protected] (K.Y.) \* Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and downregulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labeled peptide standards. We also revealed that transcription factor CEBPB and LYN kinase were involved in differentiation onset, and evaluated their protein levels by SRM technique. Obtained results indicate that the omics data reflect involvement of the DNA repair system and the MAPK kinase cascade as well as show the balance between the processes of the cell survival and apoptosis in a p53-independent manner. The differentially expressed transcripts and proteins, predicted transcriptional factors, and key molecules such as HIC1, CEBPB, LYN, and PARP1 may be considered as potential targets for differentiation therapy of acute myeloid leukemia. Keywords: acute myeloid leukemia; HL-60 cell line; ATRA; induced differentiation; transcriptome; proteome; transcription factors; key molecules; regulatory pathway modelling; SRM #### 1. Introduction Cell differentiation is a fundamental process of the development, growth, reproduction of multicellular organisms. Regulation of cell differentiation has been for decades and remains an important task for investigation due to its importance in cancer and many other diseases therapy. Leukemic cells that are induced to differentiate under all-trans-retinoid acid (ATRA) treatment make a convenient model for studying of cell maturation in vitro. Normally, ATRA in physiological dosage binds and activates a heterodimer receptor RAR/RXR followed by release of histone deacetylases (HDACs) and transcription co-repressors (N-CoR or SMRT), and by recruitment of transcription co-activators (NcoA-1/SRC-1, CBP/p300, p/CIP, and ACTR) [1]. In turn, retinoic acid response element (RARE) containing genes, which are repressed by nonactive RAR/RXR, trigger the further cascade of molecular events leading to myeloid precursor's maturation into functional granulocytes. Various mutations impair granulocytic differentiation resulting in highly heterogeneous acute myeloid leukemia (AML), which could be cured by high dosage of ATRA. In the case Citation: Novikova, S.; Tikhonova, O.; Kurbatov, L.; Farafonova, T.; Vakhrushev, I.; Lupatov, A.; Yarygin, K.; Zgoda, V. Omics Technologies to Decipher Regulatory Networks in Granulocytic Cell Differentiation. Biomolecules 2021, 11, 907. https:// doi.org/10.3390/biom11060907 Academic Editors: Christopher Gerner and Michelle Hill Received: 23 April 2021 Accepted: 15 June 2021 Published: 18 June 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). of AML subtype M3 (French–American–British (FAB) classification), a.k.a acute promyelocytic leukemia (APL), deleterious mutation, namely balanced chromosomal translocation between chromosomes 15 and 17 t (15;17) (q24; q21), affects retinoic acid (RA) receptor gene RARα resulting in formation of dominant negative fusion protein PLM-RARA [2]. The NB4 cell line that harbors such a hallmark mutation, is used as a model to study of the APL cell biology [3]. In APL cells the transcription of RA-responsive genes is blocked due to the increased avidity of PLM-RARA/RXR for co-repressor molecules [4]. The treatment with high dosage of ATRA induces dissociation of co-repressors from PML-RARA and triggers fusion protein degradation via the ubiquitin-proteasome or autophagy pathway [5,6]. The ATRA-based regimens that are used as a first-line treatment of APL patients, induce complete remission rates of 90% [7]. Nevertheless, other types of AML are not that successfully treatable with the 5-years survival rates only about 40–45% [8]. Meanwhile, antileukemic effect of ATRA was also observed in AML (non-APL) cell models, including HL-60, THP-1, MOLM-14, HF-6, and U937 cell lines [9]. The HL-60 promyelocytic leukemia cell line is classified as AML with maturation, also referred to as AML subtype M2 by FAB classification [10]. These cells were isolated in 1977 from a patient with acute myeloid leukemia. Later it was found that, these promyelocytic cells could be induced to differentiate into granulocytes in vitro by ATRA [11]. The HL60 cell genome contains normal RARα gene, an amplified c-myc proto-oncogene and deficient of p53 gene [12,13]. Notably, deletion in the p53 gene occurs at a frequency of up to 10% in de novo AML (non-APL) cases and associated with exceedingly adverse prognosis regardless of the type of mutation (missense, nonsense, small insertions, and deletions, etc.) [8]. Being ATRA-responsive, the HL60 cell line has been used for decades as a convenient model object for cell differentiation [11,14]. Omics technologies represent powerful tools for a full-scale analysis of gene and protein expression that allow for gaining important molecular information about differentiation process, and acquiring the complete picture of the cell maturation. Thus, using HL-60 (AML) and NB4 (APL) cell lines as model systems, the complexity of differentiation processes and the diversity of pathways involved in induced differentiation at transcriptome [15–17] and proteome [18,19] levels have been demonstrated. Despite the fact that proteomics and transcriptomics alone represent the powerful techniques for investigation of ATRA-induced differentiation, the systems approach is appealing to the elucidation of molecular mechanisms. In this respect, the systems study was performed on the NB4 promyelocytic cell line under ATRA treatment (alone or in combination with arsenic trioxide (ATO)) in a time-course manner. By applying microarray technology and 2D-gel electrophoresis followed by MALDI-TOF-TOF analysis, transcription factors (TFs) and co-factors responsible for global changes in transcriptional regulation and involved in stimulation of the IFN-pathway, cell cycle arrest, and activation of signal transduction have been unmasked [3]. However, even simultaneous analysis of proteome and transcriptome differences observed in the experiment is not always sufficient to unravel regulatory mechanisms. The upor down-regulation of protein and transcript levels under ATRA treatment is often caused by previous regulatory events. Predicting transcription factors, responsible for altered gene expression, and revealing, in turn, their putative regulators, a hierarchical model of induced differentiation could be built. Therefore, a bioinformatics search for upstream regulators, including transcription factors [20], is an appropriate tool for proteome and transcriptome data interpretation. Identification and analysis of TFs and regulatory pathways responsible for altered gene or protein expression that result in the cell differentiation may contribute to identification of the mechanism(s) underlying this complex process. #### 2. Materials and Methods #### 2.1. Experimental Design The time-course studying of induced granulocytic differentiation allows obtainment of the most accurate data on molecular perturbations under ATRA treatment. Previously, several schedules of HL-60 cell harvesting after ATRA treatment have been applied in the time-course experiments [3,21]. To perform transcriptomic and proteomic profiling, we selected 24 and 96 h time points, when the molecular perturbations are prominent. To reveal the molecular onset of cell maturation at transcriptome and proteome levels, we also added the 3 h time point. For proteomic experiment we also studied the time point 48 h of treatment; during this period HL-60 cells underwent two division cycles. In our preliminary mass-spectrometry experiments we did not observe any significant changes in the ATRA-induced cell proteome within the first 2 h (compared to 0 h) after ATRA induction or at 72 h (compared to 96 h) after treatment (data not shown). For proteome analysis, we performed the ATRA-induced differentiation experiments in three independent biological replicates. HL-60 cells were harvested at 0, 3, 24, 48, and 96 h after ATRA treatment (overall 15 samples). For the transcriptome analysis, HL-60 cells were subjected to ATRA treatment in three biological replicates and were harvested at 0, 3, 24, and 96 h (overall 12 samples). For the proteome analysis, the LC-MS/MS experiments were carried out in five technical replicates per time point, and the whole-genome transcriptome analysis was performed in three technical replicates per time point. Cells harvested before ATRA treatment (time point 0 h) served as controls for both transcriptomic and proteomic profiling. The study workflow is shown in Figure 1. Figure 1. The study workflow. We applied a multi-disciplinary platform to study ATRA-induced granulocytic differentiation in a time-course manner using HL-60 cell line as a model. We combined LC-MS/MS analysis (0, 3, 24, 48, and 96 h after ATRA treatment, three bio repeats), whole-genome transcriptome analysis (0, 3, 24, and 96 h after ATRA treatment, three bio repeats), and bioinformatic search for transcription factor binding sites (TFBS) and for the key regulatory molecules. To verify the predicted regulatory networks the abundance of proteins HIC1, CEBPB, LYN, and PARP1, belonging to the designed model regulatory networks or involving in differentiation onset, were measured in time-course manner by selected reaction monitoring (SRM) using synthetic isotopically-labeled peptides as standard. #### 2.2. HL60 Cells Cultures The HL-60 human promyelocytic leukemia cells (obtained from the cell culture bank Institute of Biomedical Chemistry (IBMC), Moscow, Russia) were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM L-glutamine (all Gibco™, Paisley, UK) in a CO2 incubator under standard conditions (37 ◦C, 5% CO2, 80% humidity). ATRA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol as a stock solution at 1 mM. HL-60 cells were treated with ATRA as described in [3] and control HL-60 cells were treated with an equal volume of the solvent (ethanol). Cell differentiation was evaluated by the CD11b and CD38 expression measured by flow cytometry. At the selected time points, the cells were harvested, washed twice with PBS, transferred to 1.5-mL Eppendorf tubes, and pelleted by centrifugation at 3000× g for 15 min using an Eppendorf 5424R centrifuge (Eppendorf, Hamburg, Germany). After removing the supernatants, the cell pellets were frozen in liquid nitrogen and stored until transcriptomic and proteomic analysis. #### 2.3. Transcriptome Analysis Total RNA was isolated from the cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) at each time point studied. The quality of the extracted RNA was controlled using a Bioanalyzer 2100, RNA 6000 Nano LabChips, and the 2100 Expert standard software (all Agilent Technologies, Santa Clara, CA, USA). Approximately 0.5 μg of each RNA sample was used for cDNA preparation in the reaction of the reverse transcription performed using a Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA) according to standard protocol. The cRNA samples for all time points were labeled with Cy5-CTP (Perkin Elmer, Waltham, MA, USA) and with Cy3-CTP (Perkin Elmer, Waltham, MA, USA) for the control sample (the time point 0 h). The cRNA fragmentations and hybridizations were performed using a standard protocol with an in situ Hybridization Kit Plus (Agilent Technologies, Santa Clara, CA, USA). Data acquisition was carried out using a DNA Microarray Scanner G2505C (Agilent Technologies, Santa Clara, CA, USA). The primary transcriptome data were processed using the Feature Extraction software (version 10.1.3.1; Agilent Technologies, Santa Clara, CA, USA). Statistical data analysis by ANOVA with the p-value cut-off set at 0.05 was performed using the GeneSpring GX12.5 software (Agilent Technologies, Santa Clara, CA, USA). Thus, we prepared the lists of genes that showed more than two-fold expression difference at least at one time point studied. #### 2.4. Preparation of HL60 Cells Lysates and In-Solution Digestion with Trypsin The cell samples were lysed using ice-cold buffer (150 μL) containing 3% sodium deoxycholate, 2.5 mM EDTA, 75 mM Tris-HCl (all Sigma-Aldrich, St. Louis, MO, USA), pH 8.5 and protease inhibitors cOmplete™ (Roche, Basel, Switzerland) with subsequent ultrasonication using the Bandelin Sonopuls probe ("BANDELIN electronic GmbH & Co. KG", Berlin, Germany). The cell lysates were centrifuged for 15 min at 5000× g using Eppendorf 5424R centrifuge. The supernatants were collected, and the pellets were dissolved in 100 μL of lysis buffer, and then subjected to the second round of protein solubilization as described above. The sample protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein digestion was performed according to the protocol described in detail by Zgoda et al. [22]. Briefly, the protein sample (about 100 μg) was transferred into a clean tube and denaturation solution (5 M urea, 1% sodium deoxycholate, in a 50 mM triethylammonium bicarbonate buffer (TEAB) containing 20mM dithiothreitol (DTT) (all Sigma-Aldrich, St. Louis, MO, USA) 20 mM DTT) in volume of 20 μL was added to make the final concentration of total protein close to 5 mg/mL. Then the samples were heated for 60 min at 42 ◦C and, after cooling at room temperature, 25 μL of 15 mM 2-iodoacetamide in 50 mM TEAB was added. The alkylation reaction continued for 30 min at room temperature and the sample was then diluted up to 120 μL by 50 mM TEAB to decrease the final concentration of denaturation buffer compounds and dilute the final protein concentration close to 0.5 mg/mL. Trypsin (1 μg) was added to samples and incubated overnight at 37 ◦C. The hydrolysis was stopped by adding formic acid (to a final concentration of 5%). Samples were centrifuged for 10 min at 10 ◦C at 12,000× g to sediment deoxycholic acid. The supernatant was transferred into a clean tube. In the obtained supernatants, the total peptide concentration was determined by the colorimetric method using a Pierce™ Quantitative Colorimetric Peptide Assay kit (Thermo Scientific, Waltham, MA, USA) in accordance with the manufacturer's recommendations. The peptides were dried and dissolved in 0.1% formic acid to a final concentration of 1 μg/μL. #### 2.5. Shotgun Mass Spectrometry The peptide samples obtained were analyzed using the Agilent HPLC system 1100 Series (Agilent Technologies, Santa Clara, CA, USA) connected to a hybrid linear ion trap LTQ Orbitrap Velos, equipped with a nanoelectrospray ion source (Thermo Scientific, Waltham, MA, USA). Peptide separations were carried out on a RP-HPLC Zorbax 300SB-C18 column (C18 3.5 μm, 75 μm inner diameter and 150 mm length, Agilent Technologies, Santa Clara, CA, USA) using a linear gradient from 95% solvent A (water, 0.1% formic acid) and 5% solvent B (water, 0.1% formic acid, and 80% acetonitrile) to 60% solvent B over 85 min at a flow rate of 0.3 μL/min. Mass spectra were acquired in the positive ion mode using Orbitrap analyzer with a resolution of 30,000 (m/z = 400) for MS and 7500 (m/z = 400) for MS/MS scans. The AGC target was set at 2 × 105 and 1 × 105 with maximum ion injection time 50 ms and 100 ms for MS and MS/MS, respectively. Survey MS scan was followed by MS/MS spectra for five the most abundant precursors. The higher energy collisional dissociation (HCD) was used, and normalized collision energy was set to 35 eV. Signal threshold was set to 5000 for an isolation window of 2 m/z. The precursors fragmented were dynamically excluded from targeting with repeat count 1, repeat duration 10 s, and exclusion duration 60 s. Singly charged ions and those with not defined charge state were excluded from triggering the MS/MS scans. #### 2.6. Data Analysis The mass spectrometry data were analyzed using SPIRE pipeline [23]. The raw mass spectrometry data were converted to the mzXML format with the RawToMzXML convertor and uploaded into the SPIRE server. The experimental data were assigned to five time points (0, 3, 24, 48, and 96 h); each point included three biological- with five technical replicates. The data obtained were searched by the in-built «Composite» search engine within SPIRE pipeline using the following parameters: enzyme specificity was set to trypsin, two missed cleavages were allowed. Carbamidomethylation of cysteines was set as fixed modification and methionine oxidation was set as variable modification for the peptide search. The mass tolerance for precursor ions was 10 ppm; the mass tolerance for fragment ions was 20 ppm. Human FASTA file (September 2015) was used as a protein sequence database. The spectra identified with 90% probability were assigned to peptides. The local false discovery rate for protein identification was set bellow 0.01 (locFDR < 0.01). locFDR was calculated in SPIRE utilizing randomized or decoy database searches [23]. Label-free quantitation was performed with the use of the SPIRE software by default settings. Expression ratios and p-values were calculated based on an over-dispersed Poisson model using an empirical Bayes correction [23]. The proteins with the expression fold change > 1.5, p-value < 0.05 and CV between biological repeats < 30%, were considered as differentially expressed. The imputation of missing data has not been applied to massspectrometric results. The volcano plot was obtained using VolcaNoseR web app [24]. #### 2.7. Functional Classification of Differentially Expressed Genes and Proteins Functional analysis of differentially expressed genes/proteins was carried out using the «Functional classification» option of the geneXplain platform (http://platform. genexplain.com) with GO and PROTEOME Databases (BIOBASE) implemented as a module of the GeneXplain platform. For the functional analysis of gene groups exhibiting altered expression at the selected time points of cell differentiation, the cut-off value for the probability of random gene allocation of a gene to a particular group (Adjusted <sup>p</sup>-value) was set at 5 × <sup>10</sup>−4. Only statistically significant classification of genes according to the GO categories, describing various biological processes in cells, was taken into consideration for the functional analysis. The STRING database v.11.0 was used to retrieve the protein–protein interactions (PPIs) from the lists of DEGs of MCD group at 3, 24, and 96 h. A high confidence (0.9) score was applied. The active interaction sources were experiments and curated databases. The built-in functional enrichment analysis results according to the molecular function (GO), and KEGG pathways were used for visualization. #### 2.8. Search for Transcription Factors, Putatively Regulating Gene and Protein Expression during ATRA-Induced Differentiation of HL-60 Cells The search for over-represented transcription factor binding sites (TFBS) was performed using geneXplain platform 2.0 software packages (http://platform.genexplain.com) and TRANSFAC® database [25]. The differentially expressed genes/proteins at different time points were considered as the test sets (Yes-sets). The gene/protein that did not show any expression changes after ATRA treatment were used as a background set (No-sets). The profile used for analysis contains a collection of vertebrate non-redundant transcription factor matrices. The promoter window was selected from −1000 to +100 from the transcription start site, and only the best-supported promoters of the genes analyzed were used. The cut-off values with a threshold of p-value < 0.005 were selected to obtain high-scoring binding sites. The matrices with high over-representation of site frequency in the promoters under study versus the background promoters (ratio > 1.4) were selected for further analysis. These matrices were converted to the set of the transcription factors (TFs), which can be responsible for expression changes in the group of genes/proteins under study. #### 2.9. Generation of Regulatory Networks The identification of potential master regulators in the signal transduction network was performed using the «Regulator search» module of the geneXplain platform 2.0 software (http://platform.genexplain.com). The signal transduction network was provided by the manually curated database, TRANSPATH®. The algorithm starts from a set of TFs and performs a graph-topological search in the signal transduction network upstream of transcription factors to identify the "key nodes" that can play a crucial role in intracellular signaling from various receptors to the set of TFs identified. These key nodes may be considered as master regulators of the process studied. The following setting parameters were used: TRANSPATH® database, maximal search radius R = 10, Score cutoff = 0.2, FDR cutoff = 0.05 and Z-score cutoff = 1.0. Besides FDR, for each possible additional regulator the Score, Z-score and Ranks sum values were calculated. For the proteomic data analysis, the "Context genes" option was used for the search of key regulators. In this case, passing through the common network nodes, the nodes presented at the transcriptome data were preferentially selected. Among the overall list of regulators generated after the search, the statistically significant results were selected using the Ranks sum parameter. Thus, it was possible to find the molecules characterized by equally good "Score" and "Z-score" parameters. The "Score" parameter reflects how well a key molecule is associated with the other molecules in the database and how many molecules of the input TFs are present in the network for a given key molecule. The "Z-score" reflects how the proposed molecule corresponds to the input TFs set. The ranks sum is a combination of Score and Z-score. In other words, these "trivial" expected results attract interest as the well-known "nodes" in the network (Score) and more specific key molecules for the input sample, which are less likely to be detected as an important regulator in the case of the other TF sets used simultaneously. #### 2.10. Selected Reaction Monitoring (SRM) The standard peptides for HIC1 (LEEAAPPSDPFR), CEBPB (VLELTAENER) LYN (TQPVPESQLLPGQR), and PARP1 (TLGDFAAEYAK) were obtained using the solid-phase peptide synthesis on the Overture™ Robotic Peptide Library Synthesizer (Protein Technologies, Manchester, UK) or Hamilton Microlab STAR devices according to the published method [26]. The isotopically labeled lysine (13C6, 15N2), arginine (13C6, 15N4) or serine (13C3, 15N1) leucine (13C6, 15N1) were used for isotopically labeled peptide synthesis instead of the unlabeled lysine (TLGDFAAEYAK), arginine (VLELTAENER), leucine (TQPVPESQLLPGQR), or serine (LEEAAPPSDPFR), respectively. Concentrations of the synthesized peptides were measured by the method of amino acids analysis with fluorescent signal detection of amino acids derived after acidic hydrolysis of peptides as described in [27]. SRM experiments were performed in three biological replicates with five time points each (0, 3 h, 24 h, 48 h, and 96 h) and in five technical replicates for each time point studied. The digested samples were spiked with isotopically labeled peptide to the final concentration 50 fmol/μg of total protein. Peptide samples (2 μg) were separated on a RP-C18 column, (Zorbax 300SB-C18, 3.5 m, 150 mm × 0.075 mm, Agilent Technologies, Santa Clara, CA, USA) using the nanoflow UPLC DionexUltiMate 3000 RSLC nano System Series (Thermo Scientific, Waltham, MA, USA). Peptide separation was achieved using a linear gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 60% solvent A and 40% solvent B over 25 min at a flow rate of 0.4 μL/min. SRM analysis was performed on the QqQ TSQ Vantage (Thermo Scientific, Waltham, MA, USA) with capillary voltage set at 2100 V, isolation window was set to 0.7 Da. SRM transition details for all peptides are shown in Table S8. The results were processed using Skyline software v4.1.0 (MacCoss Lab Software, Seattle, WA, USA). The coefficient of variation (CV) of transition intensity did not exceed 25%, 12%, 12%, and 6% between technical replicates for LEEAAPPSDPFR, VLELTAENER TQPVPESQLLPGQR, and TLGDFAAEYAK, respectively. #### 3. Results #### 3.1. Transcriptome Analysis and Functional Annotation of Differentially Expressed Genes during ATRA-Induced Differentiation of HL-60 Cells To validate HL-60 cell differentiation into neutrophils, expression of surface markers CD11b and CD38 was assessed by flow cytofluorometry at 96 h after ATRA treatment prior transcriptome/proteome analysis (Figure S1). Although measurement of CD11b is the most convenient way to evaluate granulocyte differentiation, to obtain more accurate data we have used additional marker CD38 that promotes induced myeloid maturation [28]. The mean fluorescence from HL-60 cells at 96 h after ATRA-treatment increased approximately 15-fold (CD38-from 171 to 2929; CD11b-from 112 to 1726) compared to untreated control. This indicates that the granulocyte differentiation of the HL60 cell line was successful. To obtain the transcriptomic data, HL-60 cells were harvested at 3 h, 24 h, and 96 h after ATRA treatment followed by mRNA microarray profiling. A total of 14,543 gene expressions were detected at all the time points studied. Among them 159, 231, and 1449 genes with fold-change (FC) ≥2 were determined as differentially expressed genes (DEGs) at 3 h, 24 h, and 96 h after ATRA treatment, respectively (Supplemental Table S1). Further, we focused on the bioinformatics reconstruction of putative regulatory pathways for DEGs that were involved in cell differentiation according to highly validated data. We annotated the altered expression genes by the Gene Ontology (GO) database category related to the biological processes (Figure 2). Figure 2. The functional GO analysis of differentially expressed genes (DEGs) of HL-60 cells at 3 h, 24 h, and 96 h after ATRA treatment. The number of DEGs (Log2 transformed) and p-value (-Log10 transformed) are provided on the x-axis. The groups from the category of "Biological process" are on the y-axis. The threshold adjusted p-value < 10<sup>−</sup>4. The group of "myeloid cell differentiation" (MCD, GO: 0030099) is marked by red color. > Figure 2 shows the DEGs at all time points were enriched by molecules, which were assigned to the group of "myeloid cell differentiation" (MCD, GO: 0030099). The MCD group was revealed at 3 h after ATRA treatment with 22 DEGs, and then was expanded up to 24 and 81 DEGs at 24 h, and 96 h, respectively. The results of the interaction analysis by STRING (Figure S2) show that the DEGs of MCD group were enriched in their interaction with the highest confidence (0.9). The KEGG database annotation revealed mapping of the DEGs of MCD group into "Chemokine signaling pathway" at 3 h and 24 h, and into "NOD-like receptor signaling pathway" at 96 h. While the data for 3 h and 24 h suggest the cytokine signaling as one of the mechanisms of the ATRA-induced granulocytic differentiation, the results for 96 h indicate the manifestation of functions of already mature neutrophils. These observations emphasize that the bioinformatics mapping of molecules with altered expression on known signaling pathways is insufficient for a complete understanding of the regulatory events. Moreover, the earliest time point (3 h after ATRA treatment) provides transcriptomic data on the granulocytic differentiation onset. The DEGs of the MCD group at 3 h included ASB2, BCL2A1, CCL2, CCL3L1, CCL4, CCR5, CD300A, CD38, CEBPB, FGR, HES1, HNR-PLL, IL8, LRG1, LYN, RELB, TNFAIP2, BCL11A, NR2F2, PTGER2, RGS18, and SERPINB2. Among them CEBPB, CCR5, CCL4, FGR, CXCL8 (IL8), and LYN form a putative functional complex according to the STRING interaction analysis (Figure S2a). These data are of great importance for deciphering the very first molecular events of ATRA-induced granulocytic differentiation. Further, the dynamics of transcription factor CEBPB and LYN kinase was assessed by targeted mass-spectrometry approach (selected reaction monitoring (SRM)) at protein level. The MCD group genes have been used for following upstream regulators search. The lists of the MCD group genes are presented in Supplemental Table S2. #### 3.2. Proteomic Analysis and Functional Annotation of Differentially Expressed Proteins during ATRA-Induced Differentiation of HL-60 Cells Proteome dynamics is associated with cell phenotype development and its continuous observation can contribute to understanding of the cell maturation process. Previously, for systems analysis of induced granulocyte differentiation and apoptosis under ATRA/arsenic trioxide treatment starting time points of 6 h at transcriptomic level and 12 h at proteomic level were used [3]. We tried to unveil the molecular onset of differentiation. In our preliminary experiments we did not observed any significant changes in the ATRA induced cell proteome within the first 2 h after ATRA induction (data not shown). We performed proteomic profiling of HL-60 cells at 0, 3 h, 24 h, 48 h, and 96 h after ATRA-treatment. Using "Composite" search engine in the SPIRE software, we identified 1436, 1470, 1379, 1253, and 1210 proteins with (locFDR) < 0.01 at the 0, 3 h, 24 h, 48 h, and 96 h time points, respectively (Supplemental Tables S3 and S4). Mass-spectrometric data are available via the ProteomeXchange with identifier PXD006768. Based on label free quantitative analysis, 122, 169, 199, and 275 proteins were revealed as differentially expressed proteins (DEPs) (FC ≥ 1.5, p-value < 0.05, CV < 30%) at 3, 24, 48, and 96 h after ATRA treatment comparing to control (0 h), respectively. Data on label free quantitative analysis and relative expression are presented in Supplemental Table S5. The heatmap of protein expression is presented in Figure S3. The DEPs are listed in Table S5. The functional analysis of DEPs was performed in the same way as for the DEGs. The results are shown in Figure 3. Figure 3a shows that the DEPs are enriched with the proteins involved in programmed cell death and its regulation at 3 h and 96 h after ATRA treatment. The five most upregulated DEPs involved in programmed cell death at 3 h after ATRA-treatment comprise proteasome subunit beta type-2 (PSMB2, P49721), apoptosis-inducing factor 1 (AIFM1, O95831), alpha-actinin-1 (ACTN1, P12814), RNA-binding protein 25 (RBM25, P49756), and apoptosis inhibitor 5 (API5, Q9BZZ5). The top five down-regulated DEPs included 26S proteasome regulatory subunit 8 (PSMC5, P62195), alpha-actinin-2 (ACTN2, P35609), 14-3-3 protein eta (YWHAH, Q04917), CD44 antigen (CD44, P16070), and protein S100-A9 (S100A9, P06702). Figure 3. (a) The functional GO analysis of differentially expressed proteins (DEPs) of HL-60 cells at 3 h, 24 h, 48 h, and 96 h after ATRA treatment. The number of DEPs (Log2 transformed) and p-value (-Log10 transformed) are provided on the x-axis. The groups from the category of "Biological process" are on the y-axis. The threshold adjusted p-value < 10<sup>−</sup>4. The groups containing proteins regulating cell death and apoptosis are marked by red. The volcano plots show the differences in proteins abundance at 3 h (b) and 96 (c) after ATRA treatment; significantly up- and down-regulated proteins are shown as red and blue dots, respectively; names are shown for five most up- and down-regulated proteins that were annotated by GO belonging to groups "programmed cell death" and/or "regulation of cell death". > The five most up-regulated proteins at 96 h after ATRA-treatment included 26S proteasome non-ATPase regulatory subunit (PSMD1, Q99460), proteasome subunit beta type-2 (PSMB2, P49721), glucose-6-phosphate 1-dehydrogenase (G6PD, P11413), thioredoxin reductase 1 (TXNRD1, Q16881), and Na(+)/H(+) exchange regulatory cofactor NHE-RF1 (SLC9A3R1, O14745). Although these DEPs are assigned to the groups regulating cell death, they affect cell fate indirectly through metabolic effects. The 5 most down-regulated DEPs included DNA-dependent protein kinase catalytic subunit (PRKDC, P78527), Bcl-2 associated transcription factor 1 (BCLAF1, Q9NYF8), DnaJ homolog sub-family A member 1 (DNAJA1, P31689), proteasome activator complex subunit 3 (PSME3, P61289), and serpin B10 (SERPINB10, P48595). The STRING interaction analysis (Figure S4) revealed that the DEPs of group "programmed cell death" and/or "regulation of cell death" were enriched in their interaction with the highest confidence (0.9) at 3 h and 96 h after ATRA-treatment. Moreover, these proteins were mapped to the "Proteasome" pathway (KEGG database annotation) with high confidence. #### 3.3. The Workflow of Transcriptome- and Proteome-Based Regulatory Networks Design The lists of DEGs and DEPs given in Supplemental Tables S2 and S5 have been used as the test sets (Yes-sets). The control sets were formed from the transcripts and proteins with unaltered expression as described in "Materials and Methods". We performed the two-step bioinformatic analysis including: To verify the molecules that are actually expressed in HL-60 cells, we matched the list of all identified and differentially expressed genes (Supplemental Table S1) and/or proteins (Supplemental Table S5) with the elements of model regulatory networks. #### 3.3.1. The Transcriptome-Based Modeling Pathway To find TFs responsible for regulation of gene expression we performed a search for the DEGs (MCD group) transcription factors binding sites (TFBS) at each time point studied (see results in Supplemental Table S6). TFs of DEGs determined at the 3/24 h and 24/96 h time were the same in general. So, in the case of time points 3, 24, and 96 h, we have combined all putative TFs in one set in order to perform key regulator search. The upstream analysis of the combined set of TFs, which are involved in regulation of MCD group genes at the 3 h, 24 h, and 96 h, revealed the top five key molecules with the lowest "Rank sum" value. The results are summarized in Table 1. Table 1. Putative key molecules responsible for regulation of the DEGs related to the myeloid cell differentiation (MCD group) at 3, 24 and 96 h after ATRA treatment. <sup>1</sup> "Reached from TF set"—the number of the TFs from the input set (Supplemental Table S6) that is reached from the respective key molecule; <sup>2</sup> "Reachable total"—the total number of molecules that can be reached from the key molecule, independent of the input set; <sup>3</sup> "Score"—the value reflecting how well the respective key molecule is connected with other molecules in the database, and how many molecules from the input set are present in the network triggered by this key molecule, the higher value—the better suitability (threshold value > 0.2); <sup>4</sup> FDR—false discovery rate (from 1000 random input sets); <sup>5</sup> "Z-score"—the value that reflects how specific each key molecule is for the input list, the higher value—the better suitability (threshold value > 1); <sup>6</sup> "Rank sum"—composite value that reflects the impact of Score and Z-score simultaneously, the lower value—the better suitability. Further, to select the key molecules for visualization, we checked either its expressions were altered at ATRA-induced granulocytic differentiation (of primary importance), and compared FDR statistics. None of the key molecules from Table 1 were significantly changed at the transcript or protein levels. At the same time, AhR and NF-kappaB1 were the most reliable based on FDR value. Moreover, AhR and NF-kappaB1 mutually regulate each other according TRANSFAC@ database. The regulatory network triggered by AhR and NF-kappaB1 is shown in Figure 4. Figure 4. The transcriptome-based model network of regulation of MCD group DEGs during ATRA-induced HL-60 cells differentiation (the time points 3 h, 24 h, and 96 h). Legend: master regulatory molecules are represented by pink ellipses; connecting molecules considered by the graph-analyzing algorithm to find the path from the TF input list to the master molecule are represented by green ellipses; the molecules from the TF input list are represented by lilac ellipses. The colored bars around molecules show changes in the expression level. Transcript expressions are shown in blue (decreased expression) or pink (increased expression) color arrays, color intensity correlates with fold-change (FC), bars are colored if FC ≥ 2. From left to right each bar represent experimental time point (the time points at 3 h, 24 h, and 96 h and additional time points at 0.5 h and 1 h). Protein expression is shown in yellow (decreased expression) and green (increased expression) color array, color intensity correlates with fold-change (FC) of relative protein expression, bar is colored if FC ≥ 1.5, from left to right each bar represent experimental time point (3 h, 24 h, 48 h, and 96 h). According to the scheme, the key molecule AhR, apparently, causes down-regulation of proto-oncogene WT1, nuclear receptor RXRα, and transcription factor E12 (TCF3) and up-regulation of PKC zeta. AhR affects GSK3beta that regulates another key molecule, NF-kappaB1. On the other hand, NF-kappaB1 affects SIRT1 deacetylase, which inhibits the transcriptional activity of RelA/p65. NF-kappaB1 also influences GSK3beta kinase, thus performing the feedback and cross-regulation from two key molecules. The model network also shows, that the NF-kappaB1/SIRT1 tandem down-regulates PARP1 (2-fold mRNA decrease at 96 h), DNA-PKcs (3-fold mRNA decrease at 96 h), and VDR (5-fold mRNA decrease at 96 h). VDR gene have been also indirectly controlled (via CSBP1) by AhR. Furthermore, NF-kappaB1/SIRT1 up-regulates TFs c-Krox, SREBP-1a, NF-AT2A-beta, and HIC1 mRNA expression. Both NF-kappaB1 and AhR trigger the up-regulation of caspase 9. These results indicate the synergistic effect of key molecules. Notably, transcriptome-based MCD-regulating scheme included various protein kinases (ERK, JNKalpha1, MKK4, GSK3beta, CSBP1 (MK14), AKT1, JNK3alpha1, Raf-1, PDK1, MKK5, and PKCzeta). This observation suggests the significant role of MAPK pathway in the regulation of DEGs of MCD group. #### 3.3.2. The Proteome-Based Modeling Pathway In the case of proteome data analysis, we have combined TFs which may regulate the expression of genes encoding DEPs (Supplementary Materials, Table S7). The results of the key regulator molecules search for DEPs are presented in Table 2. The Top-5 key molecules with the lowest "Rank sum" value are shown. Table 2. Putative key molecules that regulate DEPs at 3, 24, 48, and 96 h during ATRA-induced differentiation of HL-60 cells. <sup>1</sup> "Reached from TF set"—the number of the TFs from the input set (Supplemental Table S7) that is reached from the respective key molecule; <sup>2</sup> "Reachable total"—the total number of molecules that can be reached from the key molecule, independent of the input set; <sup>3</sup> "Score"—the value reflecting how well the respective key molecule is connected with other molecules in the database, and how many molecules from the input set are present in the network triggered by this key molecule, the higher value—the better suitability (threshold value > 0.2); <sup>4</sup> FDR—false discovery rate (from 1000 random input sets); <sup>5</sup> "Z-score"—the value that reflects how specific each key molecule is for the input list, the higher value—the better suitability (threshold value > 1); <sup>6</sup> "Rank sum"—composite value that reflects the impact of Score and Z-score simultaneously, the lower value—the better suitability. Further, to select the key molecule for visualization, we checked either its expression was altered during ATRA-induced granulocytic differentiation (of primary importance), and compared their FDR statistics. According to our transcriptomic data, we observed a 2-fold decrease of the PARP1 levels at 96 h. At the same time, PARP1 was identified in a shotgun mass spectrometry experiment. Furthermore, this molecule represents an intermediate node in the SIRT1-mediated signal transduction in the transcriptome-based network triggered by NF-kappaB1 and AhR (see Figure 4). In addition to the five most statistically significant molecular regulators, Table 2 also includes a retinoic acid receptor NR1B1 (RARα) as the key molecule. Although the Rank sum has not included RARα in the top five molecules, it has sufficient Score, Z-Score, and FDR values. Moreover, RARα is the well-known target of retinoic acid, inducing the differentiation of HL-60 cells [29]. The proteome-based scheme of TF regulation based on the selected key molecules, PARP1 and RARα, is shown in Figure 5. This modeling pathway could demonstrate molecular synergy of PARP1 and RARα. Figure 5 demonstrates that in addition to the TFs with altered expression described previously (VDR, RXRα, and HIC1) the unique TFs were predicted using the proteomic data, including IRF7 and AML3 (RUNX2) (2.6-fold mRNA increased at 96 h), and GATA2 (mRNA reduced by 3.6- and 6.5-fold at 24 h and 96 h, respectively). Figure 5. Proteome-based model network of regulation of ATRA-induced HL-60 cell line differentiation (time points 3 h, 24 h, 48 h, and 96 h). Legend: master regulatory molecules are represented by pink ellipses; connecting molecules considered by the graph-analyzing algorithm to find the path from the TF input list to the master molecule are represented by green ellipses; the molecules from the TF input list are represented by lilac ellipses. The colored bars around molecules show changes in the expression. Transcript expression is shown in blue (decreased expression) and pink (increased expression) color array, color intensity correlates with fold-change (FC) of relative mRNA expression, bar is colored if FC ≥ 2, from left to right each bar represent experimental time point (the time points at 3 h, 24 h, and 96 h and additional time points at 0.5 h and 1 h). Protein expression is shown in yellow (decreased expression) and green (increased expression) color array, color intensity correlates with fold-change (FC) of relative protein expression, bar is colored if FC ≥ 1.5, from left to right each bar represent experimental time point (3 h, 24 h, 48 h, and 96 h). According to Figure 5, DNA-PKcs also affects IkappaB-alpha (NFKBIA): its expression is 3.2- and 2.9-fold increased at the transcriptome level at the time point 3 h. Notably, the key molecule RARα (NR1B1 on the scheme) regulates PARP1 through CBP acetylase. In turn, the PARP1-triggered network regulates RARα through the DNK-PKcs/AKT1/CASP9/CASP3/SRF/JNK1α1/pCAF loop. In the case of RAR-dependent transcription, it has been found that PARP1 functions as a co-regulator, which is required to switch the mediator complex in the active state and start the transcription [30]. The same pathway branch (PARP1/DNA-PKcs/VDR) and some TFs (HIC1 and RXRα) belong to both transcriptome and proteome-based model regulatory networks that suggests the importance of these molecules and actual involvement of the pathways in the regulation of ATRA-induced differentiation of HL-60 cells. #### 3.4. Verification of Protein Levels of HIC1, PARP1, CEBPB, and LYN During ATRA-Induced Differentiation by SRM Analysis To reveal molecules of the transcriptome- and proteome-based pathways, which are actually expressed in HL-60 cells, we have matched the list of all identified and differentially expressed genes (Supplemental Table S1) and proteins (Supplemental Table S5) with molecules in the model regulatory networks. Differentially expressed genes belonging to the transcriptome- and proteome-based modeling networks are shown in Figure 6. Figure 6. Components of transcriptome- and proteome-based model networks with altered mRNA expression under ATRA treatment. Transcription factors, TFs (predicted by TRANSFAC@ database), intermediate and key molecules (predicted by TRANSPATH@ database) with fold change ≥ 2 (p-value ≤ 0.05) at 3, 24, and 96 h is presented. > Figure 6 shows that 15 molecules, including one key molecule, five intermediate molecules, and nine transcription factors (TFs) of the transcriptome- and proteome-based model networks were characterized by the altered mRNA expression level. Transcriptional repressor HIC1 was strongly up-regulated at all time points studied suggesting its regulatory value. It is noteworthy that CASP9 and NFKBIA were up-regulated at 3 h after ATRA treatment. Transcription factors VDR and RXRA, which are intimately related to induced differentiation, were down-regulated (as well as key molecule PARP1). > Among predicted regulatory molecules we selected transcription factor HIC1 and key molecule PARP1 for measuring abundance in HL-60 cells at different time points by SRM. Next, we have compared transcriptomic and proteomic profiles during ATRA-induced differentiation. We also evaluated levels of transcription factor CEBPB and LYN kinase with altered expression at the earliest time point (3 h) by SRM. Results are shown in Figures 7 and 8. > Figure 7a,d demonstrate the trace of SRM transitions for native (above) and SIS standard (below) peptides LEEAAPPSDPFR of HIC1 protein, and TLGDFAAEYAK of PARP1 protein, respectively. The Figure 7b,c show transcriptomic and proteomic profiles of HIC1 expression. Transcription repressor HIC1 was up-regulated at 3 h and its mRNA abundance gradually increased almost 9 times to 96 h. HIC1 protein has not been identified in shotgun mass-spectrometry experiment. Using SRM technique with stable isotope labeled peptide standard (LEEAAPPSDPFR) the HIC1 abundance was detected at 24 h, 48 h, and 96 h. At these time-points its concentration was 0.63 ± 0.21 fmol/μg, 0.85 ± 0.14 fmol/μg, and 1.2 ± 0.15 fmol/μg of total protein, respectively. The HIC1 protein level was increased approximately 2-fold (FC = 1.9, p-value ≤ 0.05) from 24 h to 96 h after ATRA treatment. Figure 7. HIC1 and PARP1 expressions at ATRA-induced granulocytic differentiation. (a) Trace of SRM transitions for native and stable isotope labeled peptide standard LEEAAPPSDPFR of HIC1. (b) Profile of transcript expression HIC1 during HL60 differentiation (fold change ≥ 2, p-value ≤ 0.05 at 3 h, 24 h, and 96 h). (c) Protein expression level of HIC1 obtained by SRM (three biological replicates) at 3 h, 24 h, 48 h, 96 h. (d) Trace of SRM transitions for native and standard isotopically-labeled peptide TLGDFAAEYAK of PARP1. (e) Profile of transcript expression PARP1 during HL60 differentiation (fold change ≥ 2, p-value ≤ 0.05 at 96 h). (f) Protein expression level of PARP1 obtained by SRM (three biological replicates) at 3 h, 24 h, 48 h, 96 h. Figure 8. CEBPB and LYN expression during ATRA-induced granulocytic differentiation. (a) Trace of SRM transitions for native and stable isotope labeled peptide standard VLELTAENER of CEBPB. (b) Profile of CEBPB transcript expression during HL60 differentiation (fold change ≥ 2, p-value ≤ 0.05 at 3 h, 24 h, and 96 h) (c) Protein expression level of CEBPB obtained by SRM (three biological replicates) at 3 h, 24 h, 48 h, 96 h. (d) Trace of SRM transitions for native and standard isotopically-labeled peptide TQPVPESQLLPGQR of LYN. (e) Profile of transcript expression LYN during HL60 differentiation (fold change ≥ 2, p-value ≤ 0.05 at 3 h and 96 h). (f) Protein expression level of LYN obtained by SRM (three biological replicates) at 3 h, 24 h, 48 h, 96 h. The Figure 7e,f show transcriptomic and proteomic profiles of PARP1 expression. PARP1 was selected as a key molecule for the proteome-based model network. At the transcriptome level we revealed a 2-fold decrease in PARP1 mRNA expression at 96 h. The SRM measurements for the TLGDFAAEYAK peptide of PARP1 were 13.28 ± 2.98, 10.83 ± 3.46 fmol/μg, 9.57 ± 2.88 fmol/μg, 8.28 ± 0.35 fmol/μg, and 8.77 ± 0.54 fmol/μg of total protein at 0, 3 h, 24 h, 48 h, and 96 h after ATRA-treatment, respectively. The PARP1 protein level was 1.5-fold (p-value ≤ 0.05) down-regulated by 96 h after ATRA treatment. Figure 8a,d demonstrate the trace of SRM transitions for native (above) and SIS standard (below) peptides VLELTAENER of CEBPB protein, and TQPVPESQLLPGQR of LYN protein, respectively. Figure 8b demonstrates that CEBPB was up-regulated starting from 3 h (FC = 3.6, p-value ≤ 0.05) up to 96 h (FC = 5.95, p-value ≤ 0.05) at transcriptome level. Using SRM, we measured CEBPB in amount of 1.2 ± 0.12 fmol/μg, 1.36 ± 0.31 fmol/μg, 1.98 ± 0.59 fmol/μg, 1.78 ± 0.28 fmol/μg, and 2.17 ± 0.21 fmol/μg at 0, 3 h, 24 h, 48 h, and 96 h after ATRA-treatment, respectively (Figure 8c). Figure 8e,f show transcriptomic and proteomic profiles of expression of LYN kinase. Transcriptomic data demonstrates significant LYN up-regulation at 3 and 96 h. The unique peptide (TQPVPESQLLPGQR, 21-34aa), which has been used for SRM analysis, is the LYN isoform B-specific and is mapped to the region that distinguishes isoform A from isoform B. Highresolution annotated MS2 spectrum of LYN isoform B-specific peptide TQPVPESQLLPGQR is shown in Figure S5. Protein LYN expression was detected in amount of 1.12 ± 0.2 fmol/μg, 0.8 ± 0.21 fmol/μg, 1.8 ± 0.46 fmol/μg, 2.18 ± 0.6 fmol/μg, and 2.49 ± 0.23 fmol/μg of total protein at 0, 3 h, 24 h, 48 h, and 96 h after ATRA-treatment, respectively. We observed coordinate increase or decrease at the transcript and protein level for HIC1, CEBPB, LYN, and PARP1; this confirms involvement of corresponding genes in the ATRA induced HL60 differentiation. The targeted mass-spectrometric data have been uploaded into PASSEL repository (dataset PASS01678). #### 4. Discussion Omics techniques provide a massive amount of data on the molecular state of the biological object studied. Nevertheless, in high-throughput transcriptome and proteome profiling, we always register only certain molecular consequences of regulatory events that occurred in the past (e.g., induction of the expression of the corresponding gene). Especially, proteomic research of differentiation onset is complicated by the fact that observed changes in protein levels take time. Thus, up-stream regulator search provides bioinformatics reconstruction of the molecular events up to one or several trigger points. Consistent with this, our whole-genome transcriptome results indicated activation of myeloid differentiation, whereas proteomic data demonstrated the involvement of the apoptosis pathways under ATRA treatment. However, knowing the expression differences alone does not allow us to reveal the effector that leads a biological system towards the particular molecular state. Applying up-stream regulator search and visualizing its result, we provide the putative "molecular scenarios" of how a dozen regulatory molecules decided the fate of hundreds of proteins and transcripts. After ATRA treatment leukemic cells, of which the phenotype is generally driven by genetic abnormalities, acquire features of mature granulocytes. As in the case of many others malignancy, HL-60 cells harbor genetic aberrations including the most frequent mutations: extensive deletion of the p53 gene, amplification of MYC oncogene, and monoallelic deletion of granulocyte–macrophage colony stimulating factor (GM-CSF) [11,12]. Considering this, we suggest that our model regulatory networks represent a putative way to overcome the effect of these mutations. Proto-oncogene MYC plays a crucial role in the regulation of cell proliferation, differentiation, and apoptosis [31,32]. From 16- to 32-fold MYC gene amplification in the HL-60 genome has been reported [33]. Although the decreased expression of MYC is not sufficient for triggering differentiation of HL-60 cells, it is accompanied by the inhibition of cell growth [34]. In our study, we observed a 7-fold decrease of MYC mRNA expression during granulocytic differentiation. Notably, TF MAX that binds MYC protein for activation of target genes [35] is the part of our proteome-based model network. Thus, the modeling scheme presented in Figure 4 could represent a way to overcome the deleterious effect of MYC gene amplification. Normally, the p53 gene is a crucial component of the molecular response to different kinds of cell stress including DNA damage. Namely, p53 is involved in mismatch repair, DNA double-strand break repair, and nucleotide excision repair that could accompany uncontrolled proliferation [36]. Poly(ADP-ribose) polymerase 1 (PARP1), the key molecule of the proteome-based model network, has intricate interplay with p53 in regulation of cell death and survival. PARP1 affects p53 transcriptional activity, and promotes its oncosupressive function [37]. In turn, the p53 expression level is prominently increased after DNA damage in PARP1-defiecint cells that leads to apoptosis [38]. Moreover, in the case of the multidrug-resistant leukemia cell line HL-60[R] the PARP1 mRNA expression level was up-regulated [39]. At the same time, a branch of components PARP1/DNA-PKcs/VDR, which is presented both the in transcriptome- and proteome-based model pathways (Figures 4 and 5), regulates DNA repair [40,41]. Thus, the proteome-based model network could represent a molecular bypass to overcome consequences of p53 deletion. It may be assumed that inhibition of PARP1 in p53-deficient HL-60 cells could have the similar antiproliferative effect as on BRCA1-deficient cancer cells of solid tumors [42]. This assumption is in agreement with the fact that primary blasts from patients with acute myeloid leukemia are sensitive to PARP-inhibitor Olaparib [43]. In our study SRM measurements show a trend of the diminution of PARP1 protein abundance, while the mRNA level was significantly down-regulated, 2-fold, to 96 h after ATRA-treatment. Considering the moderate modulation of abundance it is conceivable that PARP1 is regulated by post-translation modification. Figures 4 and 5 demonstrate that PARP1 could be acetylated by CREB-binding protein (CBP) or deacetylate by SIRT1. Both PARP1 and SIRT1 compete for the common NAD+ substrate and modulate each other's activity by mutual modification [44]. PARP1 inhibition by SIRT1 could contribute to the increase in the DNA damage level and cell death in the absence of p53 expression. SIRT1 stimulation by pharmacological agents could promote PARP1 inhibition. On other hand, SIRT1 can activate apoptosis by direct deacetylation of the RelA-p65 subunit that inhibits the transcription of NF-kappaB and increases cell sensitivity to TNF-alpha-induced apoptosis [40]. TNF-alpha is known to cause p53-independent apoptosis, which promotes the monocytic differentiation of HL-60 cells [45]. At the same time, we observed prominent up-regulation of transcriptional repressor HIC1 that suppresses SIRT1 gene expression. SIRT1 deacetylates and inactivates both p53 and PARP1; HIC1 affects cell cycle, apoptosis, and DNA repair. According to our transcriptome-based model network (Figure 4), HIC1 was triggered by NF-kappaB via SIRT1 and p300. In Figure 5, a proteome-based model network represents HIC1 regulated by cascade triggered by PARP1 through DNA-PKs, AKT, and p300. This suggests a feedback loop involved in maintaining moderate inhibition of SIRT1 via HIC1 that sustains PARP1 activity, resulting in delayed apoptosis and allowing cells to differentiate into neutrophils. Apparently, accumulation of critical amount of HIC1 causes SIRT1 suppression, and further PARP1 down-regulation occurs due to apoptosis-driven cleavage. It seems that the cell machinery involved in the response to the DNA damage plays a key role in induced granulocytic differentiation, and its component could be sensitive to target treatment. The transcriptome analysis provides biological data on ATRA-induced granulocytic differentiation at the whole genome-scale. However, not all transcripts detected could be traced at the protein level. In turn, despite the proteomic data being limited by the sensitivity of mass-spectrometry, the protein expression underlies the cell phenotype manifestation. As expected, different inputs to up-stream regulator search resulted in different key molecules in transcriptome- and proteome-based modeling pathways. Still, the schemas show common predicted transcription factors (SRF, ARNT, RXRA, VDR, and HIC1), intermediate molecules (Caspase9, histone acetyltransferase p300, protein kinases ERK1, Raf-1, AKT1, CSBP1 (MK14), JNKaplha1, and AKT), and even whole branches of molecular events (axis PARP1-DNA-PKcs-VDR). The gene transcription and protein synthesis are separated in time, and the above observations suggest different key regulation, but we also observe the general molecular consequences, such as the involvement of the DNA repair system and the MAPK kinase cascade. Interesting but conflicting results were obtained for LYN kinase. The previous studied demonstrated that constitutively activated LYN was involved in AML pathogenesis and treatment of cells by LYN siRNA resulted in the antiproliferative effect [46,47]. In our study we observed LYN up-regulation at mRNA level under ATRA treatment. SRM technique allows to distinguish different isoforms of the same protein. We used the isoform specific peptide standard to detect LYN isoform B and found it to be up-regulated at the proteome level. Previously it was reported that phosphorylation activity of Lyn isoform B was lower than that of Lyn isoform A [48]. Moreover, the ratio of Lyn isoform A and Lyn isoform B splice forms may represent a biomarker of neoplasm aggressiveness as was shown in the case of breast cancer [49]. Absolute quantification by SRM with SIS peptides demonstrates the almost equimolar abundance of TF CEBPB and Src kinase LYN. Considering their possible interaction (STRING analysis of DEGs, Figure S2), absolute abundances of CEBPB and LYN suggest protein stoichiometry in the putative complex involved in the earliest step of ATRA-induced granulocytic differentiation. The myeloid-associated TFs (RARa, RXR, VDR, CEBPB, and GATA2) of model schemes confirm the biological relevance of bioinformatics modeling. Notably, a transcriptomebased MCD-regulating scheme included various protein kinases (ERK, JNKalpha1, MKK4, GSK3beta, CSBP1 (MK14), AKT1, JNK3alpha1, Raf-1, PDK1, MKK5, and PKCzeta), that is in accordance with MAPK-based mechanisms for ATRA-induced granulocytic differentiation [14]. Moreover, the current inter-platform study shows the involvement of such less associated with AML TFs as NF-ATs, SMAD3, WT1, and c-Krox, as well as ubiquitous molecules (p300, P/CAF, UBC9), which are involved in posttranslational modifications (acetylation, sumoylation, ubiqutunilation etc.). All the above observations suggest the existence of alternative, RAR/RXR transcription-independent, induced differentiation pathways. However, this assumption should be experimentally proven. #### 5. Conclusions Applying transcriptomic, proteomic analysis, and bioinformatics prediction we have suggested a hypothesis on molecular mechanism of ATRA-induced granulocytic differentiation. We aimed to trace dynamics at different molecular levels in a time-course manner. The novelty of the approach used in our study is that molecules with altered expression from omics experiments have not been just mapped to known signaling pathways. Instead, an upstream regulator search aimed to obtain the hierarchical model of ATRA-induced granulocytic differentiation that reconstructs the molecular events affecting differentially expressed mRNA and proteins. Only the TFBS in the promotor region of genes with altered expression and highly validated data on protein–protein interaction were taken into account in upstream regulator search. The resulting modeling schemas are visualizations of the most probable variant of a biological signal transmission, which leads to a change in the expression levels of transcripts and proteins, observed experimentally. The validation of bioinformatics prediction by functional molecular research is an important item, and a subject of our further work. The TF HIC1 and the key molecule PARP1 are contemplated as the most promising targets for validation of the modeling pathways. The approach combining transcriptomic, proteomic analysis, and computational analysis described here is applicable to various cells models including primary blast cells from patients under different treatment regimens. Thus this platform could be useful for the goals of precision medicine such as monitoring response to treatment especially in case of drug resistance. Our results suggest that the multi-disciplinary platform combining transcriptomics, proteomics, and bioinformatics is a promising approach to reveal regulatory molecules that are hardly detected by convenient omics methods or laborious to derive from convoluted proteomic or transcriptomic data. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11060907/s1, Figure S1. Evaluation of HL-60 cell line by the CD38 and CD11b expression level measured by flow cytometry. Figure S2. STRING interaction analysis of differentially expressed genes (DEGs) of Myeloid cell differentiation (MCD) group at 3, 24, and 96 h after ATRA treatment. Figure S3. Heatmap of protein expression during HL-60 cell line differentiation. Figure S4. STRING interaction analysis of differentially expressed proteins (DEPs) assigned to group "programmed cell death" and/or "regulation of cell death" at 3 and 96 h after ATRA treatment. Figure S5. High-resolution annotated MS2 spectrum of LYN isoform B-specific peptide TQPVPESQLLPGQR. Figure S6. Calibration curves plotting of experimentally determined concentrations versus theoretical concentrations of the target analyte using isotopically labeled and label-free synthetic standard peptide A: LEEAAPPSDPFR (HIC1), B: TLGDFAAEYAK (PARP1), C: VLELTAENER (CEBPB), and D: TQPVPESQLLPGQR (LYN). Table S1. All transcripts detected and differentially expressed genes (DEGs) with fold-change equal or above 2 (p-value < 0.05) at 3 h, 24 h, and 96 h after ATRA treatment. Table S2. The transcriptomic test sets (Yes-sets) for pathway modeling: DEGs related to the myeloid cell differentiation (MCD) (GO: 0030099) at the 3, 24, and 96 h time points. Table S3. Data on spectral counting. Number of unique peptides, percent of coverage, number of spectra, q-values and local FDR are shown for each peptide in each time point (0, 3, 24, 48, and 96 h). Table S4. Data on spectral counting. Number of unique proteins, percent of coverage, number of spectra, q-values and local FDR are shown for each protein in each time point (0, 3, 24, 48, and 96 h). Table S5. Summary of relative expression analysis and proteomic test sets (Yes-sets) for pathway modeling. Table S6. Transcription factors (TFs) possibly regulating DEGs expression during ATRA-induced HL-60 cell differentiation at time points 3 h, 24 h, and 96 h. Table S7. Transcription factors (TFs) possibly regulating DEPs expression during ATRA-induced HL-60 cell differentiation at time points 3 h, 24 h, 48 h, and 96 h. Table S8. Transition for SRM method (QqQ TSQ Vantage (Thermo Scientific, USA). Author Contributions: Conceptualization, V.Z.; methodology, V.Z. and K.Y.; software, O.T.; investigation, I.V., S.N., T.F., O.T., L.K., and A.L.; data curation, S.N. and O.T.; writing—original draft preparation, S.N. and O.T.; writing—review and editing, S.N., O.T., and V.Z.; visualization, S.N. and O.T.; supervision, V.Z.; project administration, V.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Russian Scientific Foundation, grant number 21-74-20122. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Mass-spectrometric data are available via ProteomeXchange with identifier PXD006768. Acknowledgments: The authors are grateful for opportunity to use the flow cytometry and mass spectrometry equipment of "Human Proteome" Core Facility (Institute of Biomedical Chemistry, Moscow). Conflicts of Interest: The authors declare no conflict of interest. #### References ## Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI Yonghyo Kim 1,2,3, Yutaka Sugihara 2,3, Tae Young Kim 1, Sung Min Cho 1, Jin Young Kim 4, Ju Yeon Lee 4, Jong Shin Yoo 4, Doona Song 5, Gyoonhee Han 5, Melinda Rezeli 2, Charlotte Welinder 2,3, Roger Appelqvist 2, György Marko-Varga 1,2,6 and Ho Jeong Kwon 1,7,\* Received: 1 February 2020; Accepted: 25 March 2020; Published: 27 March 2020 Abstract: Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products. Keywords: target identification; target validation; label-free method for drugs; anti-angiogenesis; mechanism of action; receptor tyrosine kinases; curcumin; natural products #### 1. Introduction Identifying the protein targets of therapeutic natural products and deciphering the specific mechanisms of action at the molecular level are crucial steps in the development of natural products as drugs to treat human diseases [1,2]. Without the validation of targets and the cellular actions of natural products, these compounds may cause unexpected events, including adverse and toxic effects in patients. Thus, methods for the identification of protein targets and the understanding of molecular mechanisms of action of these natural products are pivotal for treating human diseases. Given the importance and necessity of deciphering these parameters, there have been several technological attempts to successfully identify the protein targets for natural products [3,4]. Widely-applied approaches include affinity-based matrices that label or tag small molecules, such as affinity pull-downs and phage display methods [5–7]. Nevertheless, these methods have limitations, such as changes in structural properties may occur upon labeling with chemical probes, or upon tagging functional groups for immobilization; biological activities of natural products may change as a result of the alterations in the chemical structure; these processes incur a high cost, and are time and labor-consuming; and difficulty in modifying small molecules due to availability of only virtual three-dimensional structures [8,9]. To overcome these limitations, new strategies have been suggested for target identification and validation using label-free methods [6] with natural products. Specifically, the targets are validated by utilizing changes in thermodynamic properties and structural stability when a natural product directly interacts with a cognate protein target [5,10]. These methods apply thermal [11], proteolytic [12,13] or oxidative stress [14] to analyze changes in the structural stability of protein targets. One of these methods using proteolytic stress for target validation is the DARTS method. DARTS is based on the principle of increased stability of a protein target upon interaction with a natural product, which makes the complex less susceptible to proteolytic effects. The conformational changes induced upon the interaction between the natural product and the target protein thermodynamically stabilize the protein structure [12,13]. Moreover, through unbiased DARTS approaches in combination with mass spectrometry (MS) analysis, quantitative MS based-proteomics is utilized to identify multiple target proteins in drug-treated versus control samples [15]. Although these label-free methods have several advantages that overcome many of the difficulties associated with labeling methods, researchers are yet to identify and validate directly in vivo interactions between natural products and their target proteins at the tissue or cellular level. Recently, matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has emerged as a new technology to analyze the distribution of a natural product in tissues by directly measuring the molecular mass of said molecule from the tissue sections. MALDI-MSI has also been adapted to investigate the interactions between natural products and protein targets ex vivo. MALDI-MSI is an analytical mass spectrometry technology that identifies ion peaks from a natural product at 25–100 μm resolution [16,17]. This platform can be utilized to either detect all ion masses within a tissue microenvironment or to perform selected-ion monitoring (SIM) of a single, specific ion mass. Furthermore, with the automated computational procedures [18,19], the data generated by MALDI-MSI can exhibit the spatial localization of all the detected natural products as a single integrated image. Accordingly, MALDI-MSI is a powerful tool for validating the interactions between a natural product and its protein targets ex vivo without any labeling probes or chemical immobilization [20,21]. With respect to absorption, distribution, metabolism, and excretion (ADME), MALDI-MSI could prove to be an effective approach to provide valuable information about the in vivo effects of label-free compounds in patients [22,23]. Accordingly, for overcoming the conventional limitations of target identification and analyzing the localization of interaction between natural products and target proteins at the tissue level, we combined the aforementioned methods into a systematic procedure and applied the same for target validation. To validate DARTS-MSI as a successful systematic strategy for target identification of natural products, we applied the same for a target-validated compound, curcumin, as a positive control natural compound. In the previous studies, curcumin was shown to directly and irreversibly bind to aminopeptidase N (APN), which plays a key role in tumor angiogenesis and proliferation, inhibiting its activity and hence angiogenesis [24]. According to a previous report, we identified a small natural molecule with antiangiogenic activity [25]. This molecule is called voacangine and is extracted from Voacanga africana, Trachelospermum jasminoides, or Tabernaemontana catharinensis. Preliminary experiments suggested that voacangine potentially inhibits angiogenesis. This effect was observed in tube formation assays in endothelial cells (ECs) and vascularization of the chick chorioallantoic membrane. Additionally, we also reported that voacangine significantly inhibited VEGF-induced chemoinvasion activity on HUVECs in a dose-dependent manner. However, its mechanistic pathways and molecular targets are still uncovered and not fully understood. Therefore, we focused on the investigation for the mode of action in voacangine as the model natural compound by applying the aforementioned systematic approach and various molecular experiments. In the current study, we investigated the mode of action of voacangine via label-free DARTS and successfully identified VEGFR2 as a target protein responsible for the observed antiangiogenic properties of voacangine in ECs. The direct interaction between voacangine and VEGFR2 was validated in vivo in animal models and also by analyzing the localization of voacangine by MSI in xenograft tumor tissue sections. In addition, sunitinib, a marketed drug inhibiting tyrosine kinases by targeting not only VEGFR2 but also other RTKs (EGFR, PDGFR, and FGFR), was selected as a reference compound for comparing the potency on angiogenesis and tumor suppression with voacangine [26–29]. The strategy of employing DARTS and MSI to identify and validate the downstream targets of a natural compound as demonstrated for voacangine in this study can streamline the general process of drug discovery and validation of protein targets for other biomolecules including natural products. #### 2. Materials and Methods #### 2.1. Materials and General Methods Curcumin (purity, ≥98%) was obtained from Sigma-Aldrich (St Louis, MO, USA). Voacangine (12-methoxyibogamine-18-carboxylic acid methylester) (purity, ≥98%) was purchased from THC Pharm (Frankfurt, Germany) [25], and the stock solution made in 100% dimethyl sulfoxide (DMSO) was stored at −20 ◦C, and diluted with the culture medium before the in vitro experiments. The working solution was freshly prepared in basal medium and the control group was treated with the same volume of DMSO as a vehicle control. Sunitinib (purity, ≥98%) was obtained from Sigma-Aldrich. Endothelial growth medium-2 (EGM-2) was purchased from Lonza (Walkersville, MD, USA). Dulbecco"=s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA). Vascular endothelial growth factor (VEGF), Tumor Necrosis Factor-α (TNF-α), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor-BB (PDGF-BB) were purchased from KOMA biotech (Seoul, Korea). The Transwell chamber system for chemoinvasion assay and Matrigel (growth factors reduced) were obtained from Corning Costar (Corning, NY, USA) and BD Biosciences (Bedford, MA, USA), respectively. Pronase and protease inhibitor cocktail tablets were obtained from Roche (Mannheim, Germany). Phosphatase inhibitor solution, Triton X-100, and dithiothreitol (DTT) were purchased from Sigma-Aldrich. Sodium chloride (NaCl), Tris, and glycine were obtained from Samchun Chemical Co., Ltd. (Seoul, Korea). Trifluoroacetic acid (TFA), high-performance liquid chromatography (HPLC)-grade methanol (≥99.8%) and the matrix compound, α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich, and liquid chromatography–mass spectrometry (LC–MS) hypergrade acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). Primary antibodies of phospho-VEGFR2, VEGFR2, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor α (PDGFRα), platelet-derived growth factor receptor β (PDGFR β), phospho- extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), ERK1/2, phospho-protein kinase B (Akt), Akt, APN, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-III-tubulin was purchased from Millipore (Temecula, CA, USA). Anti-epidermal growth factor receptor 1 (EGFR1) and anti-voltage-dependent anion-selective channel 1 (VDAC1) were purchased from Abcam (Cambridge, UK). Anti-fibroblast growth factor receptor 5 (FGFR5) was purchased from Thermo Fischer Scientific (Waltham, MA, USA). Anti-cluster of differentiation 31 (CD31) was purchased from Novus (Littleton, CO, USA). Secondary antibodies of anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were purchased from Cell Signaling Technology. U87 glioblastoma cells (U87MG), human umbilical vein endothelial cells (HUVECs), and human hepatoma cell (HepG2) were purchased from Korea Cell Line Bank, Seoul, Korea. #### 2.2. In Vivo Mouse Tumor Xenograft Assays Mice were housed in the pathogen-free facility of the Laboratory Animal Research Center in Yonsei University, Seoul, Korea. The mice were handled following the Institutional Animal Care and Use Committee (IACUC) (permission number: IACUC-A-201407-254-01, IACUC-A-201503-213-01, IACUC-A-201602-149-02, and IACUC-201603-422-01) and International Guidelines for the Ethical Use of Animals. U87MG cells (5 <sup>×</sup> 106 cells) suspended in 200 <sup>μ</sup>L phosphate-buffered saline (PBS)/Matrigel (1:1) were subcutaneously implanted into the dorsal flank of athymic nude mice (4-week-old female BALB/c nude mice, Orient Bio, Seoul, Korea). Once the tumors became palpable (50–100 mm3, ~2 weeks), mice were randomly selected and separated into four groups (6 mice per group), and intraperitoneally treated with vehicle, curcumin (60 mg/kg), and voacangine (10 mg/kg) daily. Sunitinib was administered orally (40 mg/kg) daily. Vehicle and drug solutions were prepared in saline:ethanol:Tween-80 (97.8:2:0.2). Tween-80 was used to enhance drug solubility. Tumor volume and mouse body weight were measured daily using the following formula: π/6 × length × width × height. Four hours after the last treatment (on day 12), mice were sacrificed, and tissue samples (tumors, livers, and kidneys) were obtained. The tissues were surgically removed and slowly frozen by placing tumors for 2 min on a plastic boat floating in a bath of isopentane that was supercooled with dry ice (−70 ◦C) [30]. All animal study protocols were performed following the Guidelines for Animal Experiments and were approved by the Department of Institutional Animal Care and Use Committee, Yonsei University, Seoul, Korea. #### 2.3. Growth Factor-Induced Chemoinvasion Assays To determine the invasiveness of HUVECs in vitro, a Transwell chamber system with polycarbonate filter inserts containing 8.0 μm pores was used. The lower side of the filter was coated with 10 μL of gelatin (Sigma-Aldrich) (1 mg/mL), and the upper side was coated with 10 μL of Matrigel (growth factors reduced, 3 mg/mL in high-grade pure water). Voacangine was added to the lower chambers in the presence of the growth factors (VEGF, TNF-α, bFGF, PDGF-BB, and EGF, 30 ng/mL each), and HUVECs (FBS starvation for 17 h, 6 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) were placed in the upper chambers. The chambers were incubated at 37 ◦C for 16 h. The invasiveness of cells fixed with 70% methanol and stained with hematoxylin and eosin (H&E) was measured by counting the total number of cells on the lower side of the filter, using an Olympus IX70 microscope at 100× g magnification. #### 2.4. Drug Affinity Responsive Target Stability (DARTS) Assay DARTS assay was performed as previously described [13,15]. Briefly, HUVECs were lysed using 0.5% Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 10% glycerin, 1 mM DTT) containing protease and phosphatase inhibitors. The supernatant from the cell lysates containing 2–3 mg/mL total protein was incubated with voacangine at the indicated concentrations at room temperature (RT) for 1 h, followed by proteolysis with pronase (1 μg/mL per sample) for 2, 5, and 10 min at RT. For curcumin treatment, the supernatant from the membrane fraction of HepG2 cell lysates containing 1.5 mg/mL total protein was incubated with curcumin at the indicated concentrations at RT for 3 h, followed by proteolysis with 10 μg/mL pronase per sample for 2, 5, and 10 min at RT. The final concentration of DMSO was 1% in all samples. To quench proteolysis, 6× sodium dodecyl sulfate (SDS) sample loading buffer (1 M Tris-hydrochloric acid (HCl) pH 6.8, SDS 10%, glycerol 60%, bromophenol blue 0.012%, and 0.6 M DTT) was added to each sample in a 1:3 ratio, thoroughly mixed, and boiled at 100 ◦C for 5 min. Samples were analyzed by immunoblotting with primary antibodies (APN, VDAC1, β-actin, VEGFR2, FGFR1, PDGFRα, and PDGFRβ) according to the manufacturer's instructions. #### 2.5. In Silico Docking Simulation All molecular docking analyses were performed with Discovery Studio 2016 software adopting the CHARMM (Chemistry at Harvard Macromolecular Mechanics) force field. The crystal structure of human VEGFR2 (Protein Data Bank code, 4AGD) was obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank. The protein structures of VEGFR2 were optimized by the Powell algorithm to minimized energy. To dock the ligands, the Ligandfit docking method was used. The parameters of Ligandfit were validated using the ligand from the VEGFR2 crystal structure. Voacangine was docked to the binding site of the protein and 10 poses were generated. The most predictive binding mode were determined based on various scoring functions (Ligscore1\_Dreiding, Ligscore2\_Dreiding, PLP1, PLP2, PMF, and DOCK\_SCORE), and the binding energies were determined by calculating the binding energy of the most predictive binding mode. #### 2.6. Immunoblotting Analysis Cell lysates were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes using standard electroblotting procedures. Blots were blocked and immunolabeled overnight at 4 ◦C with primary antibodies. For immunoblotting of tumor samples, sections (10 μm thickness) from tumor tissues were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer. Lysates from tumor samples were separated by 10% SDS-PAGE, and the proteins were transferred on PVDF membranes using standard electroblotting procedures. Blots were blocked and immunolabeled overnight at 4 ◦C with primary antibodies (phospho VEGFR2, VEGFR2 [31,32], phospho ERK, ERK, phospho Akt, Akt, and β3-tubulin) according to manufacturer's instructions. Then membranes were washed with TBST (Tris-buffered saline with 0.05% Tween-20,) 3 times for 10 min each, and the secondary antibody was added and incubated for 1 h at RT. Immunolabeling was detected using an enhanced chemiluminescence (ECL) kit according to the manufacturer's instructions. #### 2.7. Cell Growth Condition and Cell Proliferation Assays Cell lines were grown according to the recommendations and protocols of the supplier. All cells were maintained at 37 ◦C in a humidified 5% CO2 incubator. All cells were seeded in 96-well plates at a density of 2000 cells/well. Voacangine was added to the cells to determine their effect on cell proliferation. Cells were grown for 72 h and growth was analyzed using the 3-(4,5-dimehylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. #### 2.8. Quantification of Microvessel Density To measure the expression levels of the vascular marker CD31 in tumor sections, frozen sections were incubated with a primary anti-CD31 antibody. Frozen xenograft tumor sections (10 μm thickness) were incubated in primary antibody in 1% bovine serum albumin (BSA) overnight blocking buffer. After rinsing the primary antibody, the tumors were labeled with anti-rabbit Alexa-594 labeled secondary antibody (Invitrogen, 1:1000) for 1 h at RT and then counterstained with 4- ,6-Diamidino-2-phenylindole (DAPI). Microvessel density was measured by counting the number of positive structures in three random fields. The images were obtained using a confocal laser scanning microscope LSM 700 (Carl Zeiss, Jena, Germany) from the whole tumor tissue at 400× g magnification. #### 2.9. Compound Detection and Analysis of Drug Distribution Using MSI A MALDI LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized for compound characterization, drug detection, and tissue imaging. For the matrix, 7.5 mg/mL α-CHCA was dissolved in 50% ACN and 50% Milli-Q water (high-grade pure water) containing 0.2% TFA. For tissue drug imaging, the freshly frozen tissues were cut into 10 μm sections using a cryotome (Thermo Fisher Scientific, Waltham, MA, USA) and placed on glass microscope slides (Superfrost ultra plus). After drying the tissue for 1 h at RT, 0.5 mL of the matrix solution was deposited stepwise onto the tissue by an airbrush. To control spraying conditions, the position of the airbrush was constantly maintained. Mass spectra were obtained using the Orbitrap mass analyzer (Thermo Fisher Scientific, Waltham, MA, USA) at 60,000 resolution (at m/z 400). Tissue sections were sampled in the 150−800 Da mass range in positive-ion mode with a 50 μm raster size. The nitrogen laser was operated at 10.0 μJ with activated automatic gain control. For MS/MS, the curcumin peak observed at m/z 369.14, and voacangine peak observed at m/z 369.21 were isolated with a 1.0 Da window, and fragmented at 40% normalized collision energy with a 30 ms activation time, 0.250 activation Q and the fragment ions were scanned at a normal scan rate in the linear ion-trap analyzer. The minimum signal required for MS/MS spectra generation was 500 counts. Spectra were analyzed with Xcalibur v 2.1.0. software. Visualization of the compound and fragment ions was performed with ImageQuest software (Version 1.0.1., Thermo Fisher Scientific, Waltham, MA, USA). #### 2.10. Quantitation of the Precursor Compound For tissue quantitation, calibration curves of the drug and compounds were established in control tissue sections of the mice. Voacangine was diluted in 50% ACN containing 0.2% TFA. For each concentration, aliquots of 0.5 μL were applied to the tissue surface within the concentration range of 10 nM–1 mM. Spraying and detection conditions were identical to those used for the tissue sample analysis. The calibration curve was then used to estimate the tissue drug concentrations in in vivo-treated tumor sections. #### 2.11. Histochemical Analysis of Protein Target in Tissues and Compound Colocalization To compare the immunofluorescence staining of the target protein and voacangine localization in tissues, frozen sections were sequentially cut from each tumor. Voacangine distribution was determined in the sections using MALDI-MSI and H&E staining. Sequential sections were labeled with anti-APN (1:100), anti-EGFR1 (1:50), anti-FGFR1 (1:50), anti-FGFR5 (1:50), and anti-VEGFR2 (1:50) [31]. The primary antibody incubation was followed by incubation with a fluorescent-tagged secondary antibody of anti-rabbit Alexa-488 (Invitrogen, 1:500). Nuclei were stained with DAPI (Invitrogen). The images were obtained using a confocal laser scanning microscope (LSM 700, Carl Zeiss) at a 200× g and 400× g magnification by title scanning the whole tumor tissue. Overlapping regions in the tumor tissue between compounds (curcumin and voacangine)-MSI and immunofluorescence staining of receptor tyrosine kinases (RTKs) were quantitated by Image J, Adobe Photoshop, and Qupath software [33] (0.2.0-m1) by counting the pixels of each merged region at identical image sizes. #### 2.12. Statistical Analysis All data fitting and statistical analysis in different experimental groups are expressed as the mean ± standard deviation (S.D.) using GraphPad Prism and Microsoft Excel. The data shown in the study were obtained from at least three independent experiments. Statistical analyses were performed using an unpaired, two-tailed Student's t-test. P-values less than 0.05 were considered statistically significant (\* indicates p < 0.05, \\ indicates p < 0.01, \\\* indicates p < 0.001). #### 3. Results and Discussions #### 3.1. Validation of the Systematic Combination of DARTS and MSI for Natural Product-Target Protein Interaction Firstly, we performed a DARTS assay to validate the interaction between curcumin and APN (Figure 1a). The stability of APN significantly decreased after 2 min of treatment with pronase, but APN pretreated with curcumin before pronase treatment retained its stability. Secondly, we identified curcumin by mass spectra using MALDI-MS and MS/MS for the detection of curcumin at the tissue level (Figure S1a,b). Based on results obtained from mass spectra, quantitation of curcumin in tissue sections was conducted using MALDI-MSI. The precursor ion (curcumin, m/z 369.14) and fragment ions 1, 2, and 3 (m/z 176.08, m/z 245.08, and m/z 285.17, respectively) in the curcumin-treated tumor tissue sections were readily detected using MALDI-MSI (Figure 1b). In contrast, the precursor ion signals and fragment ions were not detected in control tissues (vehicle solution-treated tumors) (Figure S1c). Additionally, the intensity of curcumin was weaker in the liver and kidney tissue sections than in the tumor tissues of treated animals (Figure S1d). A merged image visualizing the transparent MSI signal of curcumin and the immunofluorescence image of APN was obtained (Figure 1c, red color). Notably, the highest concentrations of curcumin were observed in the tumor regions that expressed the highest concentrations of APN (yellow color in the high APN image). Through the quantitation of the merged pixel count, we observed that curcumin MSI showed high colocalization (72.65%) in the regions with the highest APN expression (Table 1). Figure 1. Validation of a systematic combination of drug affinity responsive target stability and mass spectrometry imaging (DARTS-MSI) for curcumin-aminopeptidase N (APN) interaction. (a) Analysis of direct binding of curcumin with APN in human umbilical vein endothelial cells (HUVECs) using the DARTS assay and immunoblotting. HUVEC lysates were incubated with curcumin and digested with pronase (0.1 μg/mL) at each incubation time. All images are the representative of three independent experiments. Each value represents the mean ± S.D. from three independent experiments. \* p < 0.05 versus control. Cur: curcumin-treated, APN: aminopeptidase N. Pro -:Pronase non-treated; Pro +:Pronase treated; -:Non-treated (b) MALDI-MSI images of curcumin (precursor ion, fragment ion 1, fragment ion 2, and fragment ion 3) on curcumin-treated tumor tissue. The results shown are representative of three independent experiments. Scale bar, 1 mm. (c) Comparison of curcumin MSI (precursor ion) and immunofluorescence staining of the target protein, APN, in curcumin-treated tumor tissue. The transparent MSI image of curcumin is overlaid on the immunofluorescence staining image for APN and is visualized in the merged region (red). Scale bar, 1 mm. Table 1. Comparison with the highest concentration of curcumin and the highest APN expressed regions in the tumor regions. Each value represents the mean from three independent experiments. #### 3.2. Identification of VEGFR2 as a Potent Target for Voacangine As the first step to determine the molecular mechanisms underlying the antiangiogenic effect and the signaling pathways involved in the process, the effect of voacangine on growth factor-induced chemoinvasion of ECs was investigated. HUVECs were treated with 10 and 20 μM voacangine in the presence of growth factors such as VEGF, TNF-α, bFGF, PDGF-BB, and EGF, and voacangine exhibited specific and potent suppression of VEGF-induced EC chemoinvasion (Figure 2a). In addition, a human phospho RTKs assay was also performed to further validate the inhibitory effect of voacangine on VEGF-mediated signaling activity (Figure 2b and Figure S2). The effect of voacangine on the phosphorylation of various RTKs was investigated in the HUVEC lysates. Figure 2. Voacangine specifically inhibited vascular endothelial growth factor (VEGF)-induced angiogenesis. (a) Effect of voacangine on chemoinvasion induced by various growth factors (VEGF, TNF-α, bFGF, PDGF-BB, and EGF). \\\* p < 0.001, \\ p < 0.01 versus control of representative growth factors. -: Non-treated (b) Quantitation of the results from human p-RTKs array assay in HUVECs. The images are the representative of three independent experiments. Each value represents the mean ± S.D. from three independent experiments. \\\* p < 0.001, \* p < 0.05 versus control. NT: non-treated control, Voa: voacangine-treated. #### 3.3. Validation of In Vitro Direct Interaction between Voacangine and VEGFR2 by DARTS Next, DARTS was performed to explore whether voacangine directly binds to VEGFR2. As shown in Figure 3a, the stability of VEGFR2 significantly decreased after a 2 min pronase digestion. In contrast, VEGFR2 pretreated with voacangine before pronase treatment retained stability even after 5 and 10 min. Figure 3. Voacangine specifically and directly binds to vascular endothelial growth factor receptor 2 (VEGFR2) (a) DARTS results from direct binding of voacangine to various receptor tyrosine kinases (RTKs) in HUVECs. HUVEC lysates were incubated with voacangine and digested with pronase (1 μg/mL) at each incubation time. Each value represents the mean ± S.D. from three independent experiments. \\ p < 0.01 versus control, \* p < 0.05 versus control. NT: non-treated control, Voa: voacangine-treated, Pro -:Pronase non-treated; Pro +:Pronase treated; -:Non-treated (b) In silico docking analysis using a 2D-diagram for validating the interaction between voacangine and VEGFR2 (juxtamembrane and kinase domains, RCSB Protein Data Bank number: 4AGD). Left panel, green (voacangine) is superimposed with VEGFR2 (grey). Right panel, binding motifs are illustrated with various interactions of voacangine with the ATP-binding pocket of VEGFR2. (c) Effect of voacangine on VEGF-induced VEGFR2 signaling. Protein levels were determined by immunoblotting using specific antibodies. The results shown are representative of three independent experiments. Voa: voacangine-treated; Sun: sunitinib-treated. To evaluate the binding of voacangine with VEGFR2, a docking model of human VEGFR2 based on its crystal structure (juxtamembrane and kinase domains, PDB number: 4AGD) was examined. In the virtual docking model of VEGFR2, two oxygen atoms of voacangine were found to reside in the hydrophobic pocket of VEGFR2 and form hydrogen bonds with Asn 923 and Cys 919, resulting in a high affinity and direct interaction with VEGFR2. The indole moiety of core directly interacted with the active residues in the VEGFR2 kinase domain (Leu 840, Val 848, Ala 866, and Leu 1035) via hydrophobic interactions (Pi-Alkyl interaction). The in-silico docking data suggested that voacangine directly interacts with VEGFR2 (Figure 3b). In pathological states, such as cancer development and tumor progression and other conditions exhibiting abnormal angiogenic phenotypes, growth factors such as VEGF are secreted from preexisting blood vessels to promote excessive cell growth. The secretion of VEGF by tumor cells ultimately leads to a remarkable promotion of angiogenesis [34,35]. Furthermore, VEGF-induced VEGFR2 signaling activates ERK and Akt by downstream phosphorylation of VEGFR2 kinase(s) and promotes angiogenesis by regulating the expression of the target gene, VEGF [36,37]. Accordingly, the effect of voacangine on VEGF-induced VEGFR2 activation and subsequent downstream signaling were investigated in HUVECs. Treatment with voacangine and sunitinib significantly suppressed the VEGF-induced phosphorylation of VEGFR2 and the downstream activation of ERK and Akt in a dose-dependent manner (Figure 3c). #### 3.4. Voacangine Inhibits Xenograft Tumor Growth and Angiogenesis In Vivo Validation of voacangine as a new VEGFR2-targeting antiangiogenic and antitumor compound was achieved by investigating its effect on tumor growth in the U87MG cell glioblastoma xenograft mouse model. U87MG glioblastoma cells form aggressive angiogenic solid tumors that exhibit high levels of VEGF and VEGFR2 [38]. As a reference, data were compared with those for sunitinib (SU11248, Sutent), a known VEGFR2-targeting anticancer drug. Both voacangine and sunitinib significantly inhibited tumor growth (Figure 4a) in 6 to 12 days without causing overt toxicity, as no significant weight loss was observed in the mice (Figure S4). As shown in Figure 4b, the expression levels of the blood vessel marker, CD31, were significantly lower in tumor-bearing mice treated with either voacangine (47.3%) or sunitinib (50%) than that of control. Figure 4. Voacangine inhibits xenograft tumor growth and angiogenesis in vivo. (a) Representative images of U87MG tumor xenograft on 12 days. Athymic nude mice bearing glioblastoma tumors consisting of U87MG glioblastoma cells were treated with vehicle, voacangine (10 mg/kg, intraperitoneal treatment), or sunitinib (40 mg/kg, oral treatment). \\\* p < 0.001 versus vehicle treatment. (b) Effect of voacangine or sunitinib treatment on the expression levels of the vascular marker, CD31 in tumor tissues. All images shown are representative of three independent experiments. White arrows indicate CD31 expression. Original magnification of fluorescence images for CD31 staining: 400× g. Scale bar, 50 μm. Microvessel density was measured by counting the number of CD31-positive structures in three random fields. Each value represents the mean ± S.D. from three independent experiments. \\\* p < 0.001 versus vehicle treatment. Veh, vehicle-treated; Voa, voacangine-treated; Sun, sunitinib-treated. High-resolution histological inspection revealed a cellular presentation within these tumors that showed a high degree of heterogeneity in cell size and shape. The sunitinib dosage (40 mg/kg, oral treatment) administered in these studies was selected from the dosage administered in the previous mice experiments [29,39,40]. Voacangine at a dosage of 10 mg/kg was administered at 48-h intervals for 14 days in the disease mouse model. A similar tendency of stabilized tumor growth was observed in mice treated with both, voacangine and sunitinib, with a fast growth onset that began on day 5, and then considerably expanded by day 8. These xenograft studies were performed with 6 animals per group, which showed a high degree of tumor growth consistency. Next, the identification of voacangine in vivo by MSI was conducted. The precursor ion (voacangine, m/z 369.2162) (Figure S5a) fragment ions 1 and 2 (m/z 309.17 and m/z 337.17, respectively) (Figure S5b) in voacangine-treated tumor tissue sections were readily-detected using MALDI-MSI (Figure 5a). In contrast, the precursor compound signal and fragment ions for voacangine were not detected in the vehicle solution-treated tumor tissues (Figure S7). Additionally, in the liver and kidney tissue sections of treated animals, the voacangine signal was detected with weaker intensity than that in the tumor tissue (Figure 5b). Figure 5. Identification of voacangine in tissue sections using mass spectrometry imaging (MSI). (a) MALDI-MSI signal for voacangine (precursor ion, fragment ion 1, and fragment ion 2) from voacangine-treated tumor tissue. The results shown are representative of three independent experiments. Scale bar, 1 mm. (b) Comparison of voacangineMSI (the precursor ion) between other voacangine-treated tumors and organ tissues (kidney and liver). #### 3.5. Validation of Target Interaction by Colocalization of Voacangine with VEGFR2 and Other RTKs As shown in Figure S8, each tumor-bearing mouse showed high VEGFR2 expression, indicating that the tumor-bearing tissues also expressed high levels of VEGFR2 which were significantly reduced by voacangine treatment. From these observations, an overlay image was visualized with a transparent MSI signal of voacangine and the immunofluorescence image of VEGFR2 in a merged region (Figure 6a, red color). Next, immunofluorescence was performed to analyze the interaction between voacangine and various other RTKs (EGFR1, FGFR1, FGFR5, and VEGFR2) in voacangine-treated tumor tissues (Figure 6b) by quantifying the pixel counts from the merged regions (Table 2). The overlaid transparent MSI image was quantitated with the immunofluorescence images of various RTKs. The merged regions are highlighted in red. Figure 6. Validation of target interaction by colocalization of voacangine with VEGFR2 and other RTKs. (a) Comparison of voacangine MSI (precursor ion) and immunofluorescence staining of the target candidate, VEGFR2, in voacangine-treated tumor tissue. The overlaid image with transparent MSI signal on the immunofluorescence staining image for VEGFR2 is visualized in the merged region (red). Scale bar, 1 mm. (b) Comparisons and quantitation of merged regions for voacangine distribution and RTK receptors (EGFR1, FGFR1, FGFR5, and VEGFR2) in tumor tissues. Regions of expression for each RTK are indicated with white dashed lines on the immunofluorescence images. The merged regions are visualized in red. The results shown are representative of three independent experiments. Each value represents the mean ± S.D. from three independent experiments. Scale bar, 1 mm. Table 2. Comparison with other RTKs in voacangine-treated tissues. Each value represents the mean from three independent experiments. IF: immunofluorescence-stained regions. Voa, voacangine. #### 3.6. Discussions Natural products have been widely used as pharmacological or nutraceutical agents for effectively treating various human diseases due to their significant biological activities with diverse chemical structures. Given the advantages of natural products, there have been many obstacles to address the exact mode of action and in vivo effects on possible target proteins. Most often these problems arise due to difficulties in chemical modification of these natural products and alteration in the chemical and biological properties during these processes. In this study, we attempted to overcome these hurdles by developing a new systematic procedure to identify targets of label-free natural products in vitro and in vivo with DARTS and MALDI-MSI, respectively. In Figure 1 and Table 1, curcumin pretreatment significantly protected APN digestion from pronase even at 10 min (p = 0.0185); but the digestion of other proteins, VDAC1 and β-actin remained unaffected, suggesting that curcumin specifically binds to APN, as reported by the earlier studies [24]. Further, these results strongly suggested that there is a stronger curcumin-binding and localization in the tumor tissue with elevated levels of APN expression. From these observations with curcumin, we suggest that the combination of DARTS and MALDI-MSI could be used as a systematic procedure for validation of in vivo target interaction with label-free natural products. After validating the interaction of curcumin with APN as a proof of concept case, we utilized this approach for voacangine, a natural antiangiogenic compound, without any known targets. As shown in Figure 2a, HUVECs were treated with 10 and 20 μM voacangine in the presence of growth factors such as VEGF, TNF-α, bFGF, PDGF-BB, and EGF, and voacangine exhibited specific and potent suppression of VEGF-induced EC chemoinvasion. These results demonstrated that voacangine did not show inhibitory activity against the other growth factors but specifically inhibit VEGF-mediated signaling. In VEGF-induced conditions, voacangine specifically suppressed the phosphorylation of VEGFR2. This was further validated by the p-RTK array assay, wherein the phosphorylation profiles of various RTKs were analyzed. Furthermore, from the 12 RTKs activated by serum out of the 45 RTKs, voacangine specifically inhibited the VEGF-induced phosphorylation of VEGFR2 (Figure 2b, Figure S2). Accordingly, the following experiments on the activity of voacangine focused on VEGFR2. Using DARTS technology, VEGFR2 was identified as a cellular target protein of voacangine. Notably, pretreatment with voacangine resulted in limited VEGFR2 digestion upon pronase treatment; however, the digestion of other RTKs, FGFR1, PDGFRα, and PDGFRβ remained unaffected, suggesting that voacangine specifically binds to VEGFR2, but not other RTKs (Figure 3a). The in vitro inhibitory activity of voacangine significantly inhibited the phosphorylation of VEGFR2 and downstream signaling proteins, such as ERK and Akt, in HUVECs (Figure 3c). These results demonstrated that the antiangiogenic mechanisms of voacangine action affect the VEGFR2 mediated signaling pathway. These also have been well-known that VEGF-mediated signaling plays a key role in tumor angiogenesis [34,35,41], and secreted VEGF binds to VEGFR2 that is expressed on the vascular endothelium. Subsequently, an angiogenic response is evoked that leads to the activation of VEGFR2 signaling [41]. VEGF-stimulated VEGFR2 induces the phosphorylation of downstream signaling kinases, including ERK and Akt which promote migration, proliferation, invasion, adhesion, and tube formation in ECs [36,37]. Therefore, targeting VEGFR2 is considered a promising strategy and an important therapeutic approach for treating diseases associated with angiogenesis, such as cancer [42]. Further evidence revealed that the effects of voacangine significantly correlated with the levels of VEGFR2 expression in different cell lines. The expression of VEGFR2 in HUVEC, U87MG, and Panc-1 cells was higher than that in the remaining cell lines (Figure S3a). As shown in Figure S3b, voacangine significantly inhibited cell proliferation in HUVEC, U87MG, and Panc-1 cells (Table S1). From the examination of the effects of voacangine on several cell lines with different VEGFR2 expression levels demonstrated that voacangine exerted its biological effects by specifically inhibiting proliferation in cells with high levels of VEGFR2 expression. Further, these results suggested that voacangine could be used as a specific inhibitor targeting VEGFR2-overexpressing cells (Figure S3). In the U87MG xenograft tumor mouse model, where VEGFR2 is highly expressed, voacangine significantly suppressed tumor growth and microvessel density in vivo without significant toxicity (Figure S4). The outcome of voacangine treatment suggested that it reduced the tumor growth to a basal level (as determined in the control groups), similar to treatment with sunitinib, which returned the tumors to baseline levels during the 14 days (Figure 4). Notably, this observation suggests that voacangine may cause dual inhibition of tumor angiogenesis and tumor growth, thus resulting in a potent antitumor activity. These results also demonstrated that voacangine might be a promising candidate for effective treatment of aggressive glioblastomas which are resistant to various chemotherapies [43]. Additionally, these data are in accord with the effects of the known VEGFR inhibitor, sunitinib. Based on these results, voacangine inhibited angiogenesis and tumor proliferation in vivo by directly targeting VEGFR2-overexpressing cancer cells, similar to sunitinib. To further validate VEGFR2 as the target receptor of voacangine, the interaction was analyzed in vivo. We established a novel systematic combination to compare the localization of small molecules and their protein targets using MALDI-MSI and immunofluorescent staining. From the MALDI-MS analyses, voacangine was identified at m/z 369.21, as well as its two major fragment ions were identified (Figure S5). On the surface of the tissue sections, a single droplet of voacangine could be detected by MALDI-MSI (Figure S6). The concentration of the voacangine and the precursor ion signal intensity linearly correlated from 0.01–100 μM. Voacangine precursor ions and its fragment ions were detected and colocalized with the tumor tissue from voacangine-treated mice. Furthermore, voacangine precursor ions and its fragment ions were not detected in control tissues, confirming that voacangine and the fragment ions observed by MALDI-MSI did not originate from the matrix or tissue. A comparative analysis of MALDI-MSI of voacangine ions and immunofluorescence images of other RTKs (EGFR1, FGFR1, FGFR5, PDGFRα, and PDGFβ), revealed that the highest concentrations of voacangine were observed in the tumor regions that expressed the highest concentrations of VEGFR2. This suggested a stronger voacangine-binding and localization in tumor tissues with elevated VEGFR2 expression (Figure 6). In a previous study, high concentrations of sunitinib colocalized with high expression levels of VEGFR2 [31]. Akin to the marketed drug sunitinib, these results demonstrated that voacangine directly interacts with VEGFR2 and therefore, can potentially be considered as a promising natural compound for suppressing angiogenesis and tumor growth by targeting VEGFR2. Among VEGFR2-targeting drugs, sunitinib is the most widely used drug for treating cancer patients [26]. Sunitinib is a multi-targeting drug and a receptor tyrosine kinase inhibitor that inhibits signaling via key angiogenic receptors, including VEGFRs, PDGFRs, and FGFRs [27]. In this study, the newly identified antiangiogenic small molecule, voacangine, was shown to interact specifically with VEGFR2. Voacangine treatment resulted in a decrease in VEGFR2 kinase activity in vitro (Figure 2b and Figure S2) and reduced its expression levels in vivo (Figure S8). Recently, many reports have demonstrated that sunitinib directly targets VEGFR2 to inhibit cancer progression in patients [44,45]. It is specifically administered as a first-line treatment to patients with advanced renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumors [46,47]. Despite the significant benefits of sunitinib treatment related to progression-free survival and disease stabilization in patients, almost all patients acquire resistance to sunitinib and relapse [48,49]. Approximately, 70% of patients show an initial response, while the remaining 30% show primary (intrinsic) resistance. Furthermore, 70% of patients acquired extrinsic resistance within 6–15 months. To treat patients with sunitinib resistance, the development of new VEGFR2 inhibitors with distinct structures and pharmacological activities is imperative for improved cancer therapy. As a possible drug candidate targeting VEGFR2 kinase, voacangine significantly inhibited in vitro and in vivo angiogenesis by directly and specifically interacting with VEGFR2. Hence, further development of voacangine as a new scaffold compound targeting the VEGFR2 kinase could provide a new option to treat cancer patients with resistance to sunitinib. Additionally, identifying and developing drug replacements from natural products such as voacangine will potentially reduce unpredicted and adverse side effects, and provide a promising strategy to improve the efficiency of small molecules in preclinical and clinical stages. #### 4. Conclusions Here, we demonstrate an effective and novel systematic combination method, consisting of a label-free method for target identification of natural products and their in vivo validation with information on "on-target" effects and bioimaging data consisting of molecular interactions in tissue samples (Figure 7). This combinatorial technique is effective not only for voacangine but could also be effectively used for many tricky natural products and could boost target identification and hence, drug development. This study provides a new systematic approach to overcome many of the problems associated with currently available methods used for in vitro and in vivo target identification and validation. Our study represents a new means to identify and validate protein targets of natural compounds as "cold compounds" and eases the exploration of the mode of action of these natural products in vitro and in vivo without any chemical modifications. These results also provide new insights into the evaluation of drug actions in tissues and the colocalization of drugs and their respective targets in vivo. Figure 7. Summary of the study. The systematic approach using the combination of DARTS-MSI for in vitro and in vivo target identification and validation of natural products. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/4/508/s1, Figure S1: Characterization of curcumin by MALDI-MS, MS/MS, and MSI, Figure S2: Voacangine specifically inhibits VEGFR2 signaling, Figure S3: Effects of voacangine on tumor growth and angiogenesis-related to VEGFR2 expression levels, Figure S4: Body weight of in vivo xenograft model, Figure S5: Characterization of curcumin by MALDI-MS and MS/MS, Figure S6: Quantitation of a droplet of voacangine on tumor tissue sections, Figure S7: Comparison of voacangine MSI between vehicle-treated tumor tissue, Figure S8: Effect of voacangine on the expression of VEGFR2, Table S1: IC50 values of voacangine in study cell lines. Author Contributions: Y.K. and H.J.K. participated in the conception and experimental designs. Y.K. and S.M.C. performed the cell and molecular biology assays, DARTS assay, tumor xenograft mouse model assay, and MALDI-MSI experiments, and analyzed the data; Y.S., T.Y.K., and M.R. carried out the MALDI-MSI and reviewed the manuscript; J.Y.K., J.Y.L., and J.S.Y. analyzed data of DARTS assay and mass spectrometry; D.S. and G.H. performed the in silico docking analysis; C.W. and R.A. contributed in the data analysis and reviewed the manuscript; Y.K., G.M.-V., and H.J.K. wrote the paper. All authors edited and approved the final manuscript. Funding: This work was partly supported by grants from the National Research Foundation of Korea, funded by the government of Korea (MSIP; 2012M3A9D1054520, 2015K1A1A2028365, 2015M3A9B6027818, 2016K2A9A1A03904900, 2018M3A9C4076477) and the Brain Korea 21Plus Project in the Korea and ICONS (Institute of Convergence Science), Yonsei University; as well as the Berta Kamprad Foundation, Lund, Sweden. Acknowledgments: We thanks that Jae-Ho Cheong (Yonsei Medical School, Korea) kindly provided the gastric cancer cells (YCC16) for cell experiments. Conflicts of Interest: The authors declare no conflict of interest. #### References © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). ## Review Interrogating Host Antiviral Environments Driven by Nuclear DNA Sensing: A Multiomic Perspective ### Timothy R. Howard and Ileana M. Cristea \* Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA; [email protected] \* Correspondence: [email protected]; Tel.: +1-609-258-9417 Received: 12 November 2020; Accepted: 23 November 2020; Published: 24 November 2020 Abstract: Nuclear DNA sensors are critical components of the mammalian innate immune system, recognizing the presence of pathogens and initiating immune signaling. These proteins act in the nuclei of infected cells by binding to foreign DNA, such as the viral genomes of nuclear-replicating DNA viruses herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). Upon binding to pathogenic DNA, the nuclear DNA sensors were shown to initiate antiviral cytokines, as well as to suppress viral gene expression. These host defense responses involve complex signaling processes that, through protein–protein interactions (PPIs) and post-translational modifications (PTMs), drive extensive remodeling of the cellular transcriptome, proteome, and secretome to generate an antiviral environment. As such, a holistic understanding of these changes is required to understand the mechanisms through which nuclear DNA sensors act. The advent of omics techniques has revolutionized the speed and scale at which biological research is conducted and has been used to make great strides in uncovering the molecular underpinnings of DNA sensing. Here, we review the contribution of proteomics approaches to characterizing nuclear DNA sensors via the discovery of functional PPIs and PTMs, as well as proteome and secretome changes that define a host antiviral environment. We also highlight the value of and future need for integrative multiomic efforts to gain a systems-level understanding of DNA sensors and their influence on epigenetic and transcriptomic alterations during infection. Keywords: DNA sensing; IFI16; cGAS; innate immunity; protein interactions; virus–host interactions; post-translational modifications; mass spectrometry; proteomics; transcriptomics #### 1. Introduction Eukaryotic cells are relentlessly assailed by a myriad of pathogens, thereby needing to constantly evolve and expand their mechanisms for pathogen detection and host defense. During infection, pathogens bring foreign sugars, lipids, proteins, and nucleic acids into host cells. These foreign molecules can act as pathogen-associated molecular patterns (PAMPs), and the ability of the cell to detect them is critical for the initiation of host defense mechanisms and the inhibition of virus production and spread. Thus, cells utilize specialized proteins known as pattern-recognition receptors (PRRs) to detect PAMPs [1]. A common PAMP detected by host cells is the pathogenic double-stranded DNA (dsDNA) from bacteria, DNA viruses, and some RNA viruses (i.e., retroviruses) [2]. PRRs for dsDNA, known as DNA sensors, bind to the pathogenic DNA and initiate defense programs that include innate immune signaling, inflammatory responses, and apoptosis. It was long believed that DNA sensors can only function outside of the nucleus, in order to avoid recognition of self-DNA and spurious activation of immune responses. However, the majority of the known human dsDNA viruses replicate within the nucleus, thereby depositing their viral genomes in the nuclei of infected cells. Examples of nuclear-replicating DNA viruses are herpesviruses, such as herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV), and Kaposi's sarcoma-associated herpesvirus (KSHV). Herpesviruses are ancient viruses that arose hundreds of millions of years ago, having ample time to co-diverge with their hosts [3–5]. The co-evolution and co-adaptation of viruses with hosts are evidenced by the diversification of PRRs and their ligand-recognition abilities [6]. Indeed, research during the past decade has demonstrated the existence of PRRs that function in nuclear sensing of pathogenic DNA [7,8]. To date, four proteins have been shown to have the ability to perform nuclear DNA sensing—in chronological order of discovery of nuclear function: interferon-inducible protein 16 (IFI16 [9–11]), interferon-inducible protein X (IFIX [12]), cyclic GMP-AMP synthase (cGAS [13–16]), and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1 [17]). The structures of these four proteins and their currently understood mechanisms for induction of antiviral responses are illustrated in Figure 1. Each nuclear DNA sensor was shown to help to induce ifnβ expression, which in turn activates numerous critical antiviral signaling pathways in adjacent cells that aim to slow the spread of infection. Ifnβ expression is thought to rely primarily on a signaling axis involving the endoplasmic reticulum membrane protein stimulator of interferon genes (STING), although STING-independent signaling has also been proposed [18]. Activation of STING leads to the phosphorylation of TANK binding kinase 1 (TBK1), which in turn phosphorylates the interferon regulatory factor 3 (IRF3). IRF3 then dimerizes, shuttles into the nucleus, and binds to the interferon-stimulated response element upstream of ifnβ to transcriptionally activate the expression of antiviral cytokines [19–22]. IFI16 was discovered as a sensor ten years ago [9], becoming the first known nuclear DNA sensor. Both IFI16 and IFIX belong to the PYHIN family of proteins [12]. These DNA sensors consist of an N-terminal pyrin domain (PYD) [23] and either one (IFIX) or two (IFI16) C-terminal HIN-200 domains [24,25] (Figure 1A). The HIN-200 domains facilitate sequence-independent binding of the sensor to the viral DNA [25], while the PYD mediates homotypic oligomerization [26,27]. IFI16 was shown to bind incoming viral dsDNA at the nuclear periphery, immediately following the docking of the virus capsid at the nuclear pore, and the PYD was found to be necessary for the IFI16 recruitment to the nuclear periphery [15]. The IFI16 oligomerization upon binding to viral DNA and recruitment of other host factors is thought to build an antiviral scaffold capable of both activating immune signaling [9,10,26,28,29] and suppressing viral transcription [29–32] (Figure 1B). A subset of IFI16 was shown to be able to shuttle between the nucleus and the cytoplasm to function in DNA sensing in a localization-dependent manner [9,10]. However, during the early stages of infection with nuclear-replicating viruses, IFI16 does not appear to move to the cytoplasm, remaining predominantly nuclear. Thus, a still unanswered question is how IFI16 communicates with STING or whether a STING-independent mechanism also contributes to ifnβ induction. IFIX was also shown to bind dsDNA in a sequence-independent manner and to help induce antiviral cytokine expression upon herpesvirus infection [12]. Furthermore, similar to IFI16, this PYHIN protein displayed pronounced ability to undergo nuclear oligomerization via its PYD [26] and was shown to also function in suppressing viral gene expression [33]. However, very few studies have so far focused on IFIX during infection, and the mechanisms involved in IFIX-mediated antiviral responses remain poorly understood. The mechanism by which cytoplasmic cGAS induces STING activation is well defined. cGAS contains an NTase core domain (Figure 1A) that catalyzes the formation of 2- 3- -cyclic GMP-AMP (cGAMP) (Figure 1B). After binding to dsDNA, cGAS dimerizes and initiates cGAMP production. This small molecule then binds to STING, causing a conformational change and dimerization that leads to TBK1 phosphorylation. The additional presence of cGAS in the nucleus has been initially the subject of debate, although it was shown to form a functional nuclear interaction with IFI16 [14]. However, in recent years, it has become accepted that cGAS indeed has nuclear localization in different cell types, and studies have characterized mechanisms that prevent its autoreactivity [34] or that underlie its nuclear function in inhibiting DNA damage repair [16,35]. Finally, the most recently discovered nuclear DNA sensor, the heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1), has classically been understood to play a role in transporting mRNA into the cytoplasm [36,37]. In 2019, it was found that, during HSV-1 infection, hnRNPA2B1 both facilitates the export of IFI16, cGAS, and STING mRNA molecules to the cytoplasm and binds viral DNA within the nucleus, shuttles to the cytoplasm, and activates STING–TBK1–IRF3 signaling [17]. Figure 1. Nuclear DNA sensors bind to viral DNA and activate antiviral cytokine signaling. (A) Domain maps for each nuclear DNA sensor. IFI16 and IFIX belong to the PYHIN family of proteins and each contain an N-terminal pyrin domain that mediates protein interactions and one or two HIN-200 domains that bind dsDNA in a sequence-independent manner. cGAS consists of overlapping Ntase core (cGAMP production) and Mab21 (DNA binding) domains. hnRNPA2B1 possesses two RNA recognition motifs, the first of which has been proposed to also contain the DNA binding site. Each protein contains a nuclear localization signal (red bars). (B) Model for the intrinsic and innate immune activity of IFI16, IFIX, cGAS, and hnRNPA2B1. During infection, IFI16 and IFIX bind viral DNA entering the nucleus through a nuclear pore complex. After binding to viral DNA via their HIN domains (blue), these proteins each form homo-oligomers mediated by the PYD in order to build antiviral signaling scaffolds necessary for the repression of viral transcription and induction of IFNß. cGAS was shown to stabilize nuclear IFI16 levels during HSV-1 infection to promote immune signaling. In the cytoplasm, cGAS binds to foreign DNA and produces cGAMP, which in turn activates the STING–TBK1–IRF3 signaling axis to induce IFNß. hnRNPA2B1 binds viral DNA and is then demethylated by JMJD6. This is required for hnRNPA2B1 dimerization and subsequent translocation into the cytosol, where it activates the STING–TBK1–IRF3 axis. In each case, IFNß protein is secreted from the cell in order to communicate with and initiate antiviral programs in neighboring cells. The importance of these nuclear DNA sensors is highlighted by the various strategies acquired by viruses during their co-evolution with their hosts and adaptation to human cells to inhibit these DNA sensors and their antiviral functions. For example, HSV-1 promotes the degradation of IFI16 by targeting this pyrin domain. Several studies have showed this degradation to be primarily driven by the viral E3 ubiquitin ligase, ICP0 [12,15,28], while other studies suggested the contribution of other factors [38]. IFIX was also found to be degraded during HSV-1 infection, and this, yet to be discovered, inhibitory mechanism was shown not to be dependent on the ICP0 E3 ubiquitin ligase activity [33]. HSV-1 further utilizes the tegument protein pUL37 to suppress the cGAS-mediated catalysis of cGAMP through deamidation of a single arginine residue in the cGAS activation loop [39]. HCMV also acquired a mechanism to inhibit the function of nuclear sensors by preventing PYD oligomerization of IFI16 and IFIX [26]. This virus immune evasion strategy uses the major tegument protein of HCMV, pUL83, to clamp the PYD, block oligomerization, and inhibit subsequent immune signaling [26]. The mechanisms described above paint a picture of intricate signaling pathways that underlie the cellular intrinsic and innate immune systems that nuclear DNA sensors feed into and the opposing virus immune evasion strategies. On the host defense side, pathogenic DNA is bound by nuclear DNA sensors which then fulfill two roles: (1) activate immune programming and (2) suppress viral gene expression. These processes rely on interactions between biomolecules, are regulated by these interactions and post-translational modifications (PTMs) and affect the expression of hundreds of cellular and viral transcripts and proteins. Therefore, understanding nuclear DNA sensing requires a holistic approach in which all these factors are considered. Knowledge of DNA sensor mechanisms is also relevant for understanding human diseases and the development of therapies. Dysregulation of DNA sensors contributes to several autoimmune disorders. For example, patients with systemic lupus erythematosus, Sjögren Syndrome, and systemic sclerosis exhibit significantly elevated levels of anti-IFI16 antibodies [40–42], which can result from aberrant overexpression and mislocalization of IFI16 [43]. Further, autoreactivity of cGAS contributes to Aicardi–Goutières syndrome (AGS) [44,45], and small molecule inhibition of cGAS activity alleviates constitutive interferon expression in an AGS mouse model [46]. Therefore understanding mechanisms regulating DNA sensors can provide important insights into driving factors of autoimmune disorders. Targeting DNA sensors or their activated pathways is also relevant in the development of both antiviral treatments and vaccines. For example, the STING–TBK1–IFNα/β signaling axis mediates the adjuvant effects required for successful immunogenicity with plasmid DNA vaccines [21,47]. Thus, we must consider how DNA sensors upstream of interferon induction react during the administration of DNA vaccines. So far, only the cytosolic PYHIN protein absent in melanoma 2 (AIM2), which directs the maturation of proinflammatory cytokines IL-18 and IL-1β, has been demonstrated to act as a sensor for DNA vaccines [48]. Interestingly, immune responses elicited by DNA vaccines in vivo seem to be cGAS- and IRF3-independent [49]. Further investigations can help elucidate the relative contributions of these DNA sensors to aiding immune memory upon DNA vaccine administration. Omic methods have significantly contributed to the emergence of the research field of nuclear DNA sensing, helping to build the current level of understanding of the underlying molecular mechanisms. Mass spectrometry (MS)-based proteomic approaches have allowed the discovery of functional regulatory hubs for nuclear DNA sensors, including protein interactions and PTMs, as well as the monitoring of DNA sensor activation (e.g., cGAMP production). Whole-cell proteome analyses and secretome investigations have informed of global cellular changes that take place during the host activation of immune signaling cascades. Transcriptome studies have started to uncover the contribution of some of these DNA sensors to repression of viral gene expression. Here, we review findings stemming from the application of proteomics and other omic methods to characterizing the function and regulation of nuclear DNA sensors and explore the future promise of multiomic approaches in understanding human immune responses to nuclear-replicating viral pathogens. #### 2. DNA Sensor Identification and Characterization through the Lens of Proteomics The use of proteomics directly led to the discovery of all known nuclear DNA sensors. As research into DNA sensing has intensified over the past decade, proteomics studies have been crucial for examining the functions and regulations of nuclear DNA sensors (Figure 2). These investigations have focused on proteome changes, protein–protein interactions (PPIs), and PTMs connected to nuclear DNA sensors in order to uncover the mechanisms of DNA sensing in response to viral infections. Here, we discuss the main MS-based approaches used for discovering DNA sensor interactions and PTMs that contribute to either promoting or inhibiting their host defense functions during viral infections (Table 1). Figure 2. Yearly research articles investigating nuclear DNA sensors. Research papers focused on each nuclear DNA sensor, obtained from PubMed search when considering published research articles each year since 1990. The sum of each year's articles for each protein is represented by line graphs (left Y axis) while articles specifically utilizing proteomics approaches to investigate proteome changes, protein–protein interactions, post-translational modifications, etc., are shown as stacked bars (right Y axis). Of note, the black line represents the number of articles concerning all kinds of DNA sensing, including non-nuclear sensors such as the cytoplasmic AIM2 and endosomal TLR9. The red dashed line marks the discovery of IFI16 as the first nuclear DNA sensor. #### Biomolecules 2020, 10, 1591 #### 2.1. DNA Sensor Molecular Interactions Drive Host Antiviral and Virus Immune Evasion Mechanisms Affinity purification-mass spectrometry (AP-MS) has been the cornerstone of identifying and quantifying protein–protein and protein-nucleic acid interactions [58]. In this approach, either a protein of interest or DNA is isolated and the accompanying interacting proteins are analyzed using mass spectrometry. Immunoaffinity purification (IP) is carried out by using an antibody conjugated to a resin, such as magnetic beads, which can be easily separated from the cell lysate and captured via centrifugation or application of a magnet (reviewed in [59]). The antibodies used can be raised against the endogenous protein of interest. However, as the efficiency and specificity of the isolation relies on the quality of the available antibody, antibodies against tags such as FLAG, HA, and GFP are often used to facilitate protein isolation [60]. DNA can be purified from cells through similar methods, usually using biotinylated DNA and streptavidin-coupled beads to isolate DNA–protein complexes [9]. Following complex isolation, the identities and abundances of the accompanying proteins are then characterized using MS. It has long been understood that viral DNA activates innate immune responses, including ifn-β expression [61], but the identities of the DNA sensors and subsequent signaling pathways remained undetermined. AP-MS approaches have been at the core of discovering the identities of DNA sensors. IFI16 was recognized as a DNA sensor in 2010, when Unterholzner et al. performed AP-MS after transfecting THP-1 cells with a biotinylated 70 base-pair vaccinia virus DNA fragment (VACV 70mer) [9]. It is of note that IFI16 is expressed and localized to both the nucleus and cytoplasm in macrophages such as the macrophage-like differentiated THP-1 cells. Further studies demonstrated that IFI16 has DNA sensor activity in the nucleus after different types of infections with nuclear-replication DNA viruses, including HSV-1 [9,10,28], KSHV [11], and HCMV [30], as well as after retrovirus infection, recognizing DNA intermediates of human immunodeficiency virus 1 (HIV-1) [6,62]. The interaction between IFI16 and HSV-1 DNA was also demonstrated in an elegant study that utilized 5-ethynyl-2- deoxycytidine (EdC) labeling of viral genomes coupled with AP-MS to investigate temporal viral genome-protein interactions. Here, IFI16 was found to associate with the viral genome by 2 h post-infection [63]. Recently, IFI16 was identified in an AP-MS study isolating the RNA genome of Chikungunya virus [64]. This is an unexpected finding as IFI16 has no known RNA sensing capability, but it implicates IFI16 in immune sensing pathways beyond dsDNA virus infection. AP-MS was also integral in the discovery of the most recently identified nuclear DNA sensor, hnRNPA2B1, which was shown to function during HSV-1 infection [17]. In this study, HSV-1 genome biotinylation and AP-MS was integrated with a characterization of the nuclear and cytoplasmic proteomes following cellular fractionation. This allowed the authors to identify hnRNPA2B1 as a protein that both binds to viral DNA and shuttles to the nucleus to activate STING–TBK1–IRF3 signaling. As nuclear DNA sensors do not directly stimulate interferon expression, interaction with other cellular proteins is crucial for initiating immune signaling pathways. Furthermore, the importance of PPIs in the regulation of immunity is highlighted by the virus–host protein interactions through which viruses inhibit DNA sensors. Thus, IP-MS studies that define the interactomes of DNA sensors have led to a better understanding of both their action and regulation. The first interactome study of IFI16 during HSV-1 infection used AP-MS to characterize interactions with both endogenous and tagged IFI16 [50]. This study revealed IFI16 interactions with many cellular transcription and chromatin regulators, such as the upstream binding transcription factor (UBTF) and ND10 body components, as well as with the nuclear architecture proteins SUN1 and SUN2. Several viral proteins were also found to associate with IFI16 [50], including the E3 ubiquitin ligase ICP0 that was previously implicated in targeting IFI16 for degradation (Figure 3) [28]. Both UBTF and ND10 bodies (also known as PML nuclear bodies) were shown to function in host defense by repressing HSV-1 transcription [65,66], and ND10 bodies were also found to be targeted for degradation by ICP0 [67]. Figure 3. Protein–protein interactions contribute to the activation or inhibition of DNA sensor. Over the course of viral infection and immune signaling, DNA sensors interact with other cellular and viral proteins. Several of these cellular proteins are important for the function of the DNA sensors for both suppressing virus replication by repressing viral transcription and inducing antiviral cytokines. Protein interactions are also used to regulate DNA sensor function. Viruses have evolved distinct mechanisms to facilitate immune evasion and cells must also possess mechanisms to prevent excessive immune signaling. Although localized to both the nucleus and cytoplasm, protein interactions with cGAS are best characterized in the cytoplasm. Nuclear proteins are shown here as rectangles and cytoplasmic interactions as hexagons. To further clarify how these interactions are facilitated and regulated during HSV-1 infection, the domain-specific interactomes of IFI16 were investigated by performing separate IP-MS experiments for the PYD and HIN domains [15]. This study revealed that the PYD interacts with members of ND10 bodies, cGAS, and the RNA polymerase II-associated factor 1 (PAF1). More recently, IP-MS with oligomerization-deficient IFI16 mutants demonstrated that IFI16 oligomerization is needed for the formation of these interactions with PAF1 and other members of the PAF1 complex during HSV-1 infection [29]. Additional experiments uncovered an antiviral role for PAF1, showing its ability to repress virus gene transcription. Similar IP-MS interactome studies of PYHIN proteins related to IFI16 led to the discovery and characterization of IFIX as an antiviral nuclear DNA sensor [12]. At the time, very little was known about the cellular role of IFIX, but through IP-MS it was found to interact with many of the same proteins as IFI16, including ND10 body components and other chromatin remodeling and immune signaling proteins. These interactions, in conjunction with its structural similarities to IFI16, suggested that IFIX may also have antiviral properties and function in DNA sensing. Follow-up experiments demonstrated that IFIX binds viral DNA, suppresses HSV-1 replication, and induces interferon expression [12]. Probing the IFIX interactome even further during HSV-1 infection revealed associations with several components of the five friends of methylated chromatin target of Prmt1 (5FMC) complex [33], which functions in epigenetic regulation [68] and was later found to also interact specifically with oligomerized IFI16 [29]. Several important discoveries of cGAS function have been made using AP-MS, and we must also emphasize that the discovery of cGAS as a DNA sensor was initially enabled by the MS characterization of the cellular proteome. Stimulation of STING by cGAMP was discovered in 2013 [69], but the source of the cyclic GMP-AMP synthase activity remained unclear. Thus, cGAS was identified by integrating shotgun proteomics and cellular fractionation in order to pinpoint the protein whose expression pattern matched that of cGAS activity [13]. Since then, targeted IP-MS studies focused on specific interactions of interest uncovered cGAS associations with several cellular proteins that support immune function, including TRIM56 [70], PARP1 [16], and IFI16 [14], among many others (Figure 3). The interaction between cGAS and IFI16 is particularly interesting because it touches on the question of redundancy for these proteins in the nuclear DNA sensing pathway. It was determined that, during HSV-1 infection, nuclear cGAS interacts with IFI16 for the purpose of stabilizing IFI16 in order to promote immune signaling [14,71]. The knowledge of cGAS interactions was later expanded with an IP-MS study of its interactome, which was further integrated with quantitative profiling of cellular proteome alterations during HSV-1 infection [51]. This interactome revealed the cGAS interaction with the RNA sensor OASL, which was demonstrated to repress cGAS activity as a host negative feedback loop for regulating cytokine induction [51]. Currently, the only study to have utilized AP-MS to study hnRNPA2B1 in the context of DNA sensing is the one in which it was discovered [17]. As indicated above, here, biotinylated HSV-1 genomes were isolated early during infection and the interacting proteins were identified via MS. These data were then cross-referenced with shotgun MS of nuclear/cytoplasmic fractionated cells in order to identify proteins that undergo nucleocytoplasmic translocation during infection. This approach enabled the authors to identify proteins that both bind viral DNA and shuttle to the cytoplasm, potentially for the purpose of activating STING–TBK1–IRF3. IP-MS was then utilized to gain a mechanistic understanding of interferon induction by hnRNPA2B1, showing that it does indeed interact with STING and TBK1 following HSV-1 infection. The discovery of interactions with nuclear DNA sensors has also led to the characterization of mechanisms by which viruses evade cellular innate immunity. For example, recognizing the ability of the HCMV tegument protein pUL83 to inhibit the nuclear oligomerization of the pyrin domains of IFI16 and IFIX (Figure 3) derived from the identification of their interactions from an IP-MS study [26]. In agreement with its reported ability to target IFI16 for degradation during HSV-1 infection [28], the ICP0 interaction with IFI16 was demonstrated by IP-MS [50]. IP studies followed by targeted assays were valuable for identifying other mechanisms of virus immune evasion, such as the inhibition of cGAMP production by the KSHV virion protein ORF52 [72] and the HSV-1 tegument protein pUL37 (detailed in the PTM section below) [39] (Figure 3). #### 2.2. Post-Translational Modifications for Finely Tuning DNA Sensor Function Beyond interactions with other biomolecules, the ability of DNA sensors to detect and respond to pathogenic invasion is closely tied to their regulation by PTMs. Changes to protein structure via phosphorylation, acetylation, ubiquitination, and SUMOylation, among others, enable the rapid regulation of protein function, and the addition or removal of PTMs is a tightly regulated cellular process in response to stress. MS has been well-established as the main method for accurate and unbiased detection of site-specific PTMs in different cellular contexts and has also contributed to the discovery of a multitude of DNA sensor PTMs (Table 2). Broadly speaking, PTMs are inherent to the ability of a cell to induce immune signaling cascades in response to pathogen infection. The necessity of PTMs for immune signaling is exemplified by the activation of IFNβ expression that hinges upon phosphorylation of both TBK1 and IRF3 in STING-dependent signaling [1]. Further, PTMs of DNA sensors have been shown to directly contribute to immune activation. The hnRNPA2B1 interactome also revealed an interaction with the nuclear protein JMJD6, which facilitates demethylation of hnRNPA2B1 at Arg226. This alteration in hnRNPA2B1 structure is necessary for its dimerization, nucleocytoplasmic translocation, and subsequent interferon induction [17]. Thus, the necessity of Arg226 demethylation for hnRNPA2B1 DNA sensing highlights the importance of protein modification in this immune response. Table 2. Known post-translational modifications of nuclear DNA sensors. The initial discovery of IFI16 as a viral DNA sensor pointed to its ability to recognize pathogenic DNA in the cytoplasm, and further characterization of this sensor also solidified its nuclear DNA sensing function. However, the mechanisms regulating IFI16 subcellular localization remained unknown. Furthermore, its relative nuclear or cytoplasmic distribution was shown to be cell type dependent, with its localization being predominantly nuclear in lymphoid, epithelial, endothelial, and fibroblast cells, tissues that tend to be among the first infected by an invading virus. In 2012, our group reported that IFI16 contains a bipartite nuclear localization signal (NLS) and, using MS, identified several acetylation sites within the NLS [10]. IFI16 mutation experiments indicated that NLS acetylation at Lys99 and Lys128 inhibits nuclear import and abrogates IFI16 DNA sensing during HSV-1 infection. This discovery was critical for supporting that IFI16 predominantly senses viral DNA within the nucleus during herpesvirus infection. A number of studies have since demonstrated that IFI16 is regulated by different types of PTMs during viral infections, which additionally include phosphorylation and SUMOylation (Table 2) [10,73–76]. PTM-driven mechanisms also underly the ability of the cell to activate DNA sensors by modifying viral immune evasion proteins, thereby crippling their functions. For example, eight phosphorylation sites were discovered on the HCMV tegument protein pUL83 and mutational analyses demonstrated that its binding to the IFI16 PYD can be compromised by Ser364 phosphorylation within the pUL83 pyrin association domain [26]. PTMs of cGAS during DNA sensing have also started to be recognized for their importance in cGAS regulation and function, and MS-based PTM analysis has been crucial for identifying key regulatory hubs. For example, Zhang et al. found that the HSV-1 tegument protein pUL37 antagonizes cGAS during infection [39]. This protein is a known deamidase that acts on the dsRNA sensing protein RIG-I [87] to prevent immune signaling during HSV-1 infection; thus, the authors proposed a similar deamidation event would prevent cGAS signaling. Using tandem MS, they discovered several deamidation sites within the Mab21 enzyme domain and further identified that deamidation of Asn210 indeed impairs the ability of cGAS to produce cGAMP upon binding to dsDNA [39]. Several other important cGAS PTMs have been identified in recent years that function to either suppress or activate cGAS activity during DNA sensing. These PTMs include phosphorylation, glutamylation, ubiquitination, and SUMOylation (Table 2). An IP-MS study of cGAS followed by mutational analysis of the identified modified sites led to the finding that the kinase Akt phosphorylates cGAS Ser305, suppressing cGAMP production and interferon expression [78]. Additionally, glutamylation of cGAS at two distinct sites have been shown to impede cGAS activity [77]. After identifying that the cytosolic carboxypeptidases 5 and 6 (CCP5 and CCP6) contribute to activation of IRF3 during infection with DNA viruses HSV-1 and VACV, Xia et al. used MS to identify cGAS as a substrate of these protein. As CCP5 and CCP6 reverse glutamylation, this then led to the discovery that cGAS activity is suppressed through Glu302 monoglutamylation by tubulin tyrosine ligase-like protein 4 (TTLL4), which prevents cGAMP production, and through Glu272 polyglutamylation by TTLL6, which weakens the cGAS DNA binding ability [77]. More recently, MS analyses led to the discovery that cGAS is also acetylated at several lysine residues, with acetylation at Lys384, Lys394, and Lys414 suppressing cGAS-mediated cGAMP production [52] and apoptosis [53], and Lys198 acetylation promoting cGAS-induced antiviral cytokine expression [53]. Targeted MS/MS quantification of site-specific acetylation during infection demonstrated that the level of Lys198 acetylation decreased during HSV-1 and HCMV infections [53], pointing to the possible presence of a viral immune evasion strategy targeting this residue to control host immune response. Targeted studies that do not utilize MS have also identified important cGAS PTMs (Table 2). Mutational analysis of cGAS revealed that phosphorylation at Tyr215 inhibits cGAS nuclear translocation upon DNA damage, and a tyrosine kinase knockdown screen showed that B-lymphoid tyrosine kinase controls phosphorylation at this residue [16]. As another example, SUMOylation of murine cGAS by TRIM38 enhanced cGAS DNA sensing by preventing polyubiquitination and subsequent degradation of cGAS [79]. Further investigations of the aforementioned interaction between cGAS and TRIM56 revealed that TRIM56 acts to monoubiquitinate cGAS in order to promote its dimerization and facilitate cytosolic DNA sensing [70]. #### 3. Defining the Cellular Landscape Representative of Immune Activation In addition to providing specific information regarding the regulation of nuclear DNA sensors, omic studies have also informed of the global alterations occurring in host cells during immune activation. Infections with DNA viruses result in major changes in mRNA expression, protein abundances, interaction networks and PTMs, cellular metabolism, and secretion. During infection, the virus seeks to inhibit host defenses, co-opt cellular machinery, and rewire the cellular metabolome to facilitate production of progeny virions. Meanwhile, the host attempts to reduce energy expenditure while producing and secreting antiviral cytokines that will slow the spread of infection. Transcriptome, proteome, metabolome, and secretome studies have been critical for gaining an understanding of these broad cellular alterations occurring during the progression of virus infections. Temporal transcriptomic and proteomic investigations have been carried out to determine whether a regulation occurs through changes at the transcript or protein level during infection and to correlate expression trends with phenotypes. Given that viruses appropriate the host cell transcription machinery and RNA processing, a range of transcriptome studies have been performed to monitor temporal cellular and viral transcript levels during different types of infections. For example, DNA microarrays have been used extensively to study the effect infection on transcription by HSV-1 [88,89], HCMV [56,90–93], KSHV [94,95], and the porcine alphaherpesvirus pseudorabies virus [96,97], among others. Similar to proteomic technologies, improvements in sequencing methods have greatly impacted our understanding of host cell response to viral infection. The emergence of RNA sequencing (RNA-seq) as an unbiased method that is both more sensitive and precise than microarrays [98] has benefitted the fields of virology and immunology by more broadly capturing the cellular and viral transcriptional landscape during infection, including the expression of interferon-stimulated genes (ISGs). This technique was used to demonstrate that HSV-1 infection of skin fibroblasts led to the upregulation of 596 genes, downregulation of only 61 genes, and 1032 alternative splicing events [99]. RNA-seq analysis of HCMV infection in human fibroblasts showed that genes involved in the epithelial-to-mesenchymal transition (EMT) are downregulated, while genes that support mesenchymal-to-epithelial transition (MET) are induced, suggesting HCMV prefers an epithelial cellular state for replication [100]. Furthermore, RNA-seq has recently been used to explore transcriptomic differences between endemic Kaposi's sarcoma (EnKS) and epidemic Kaposi's sarcoma (EpKS), which results from KSHV and HIV-1 co-infection in sub-Saharan Africa [101]. This study found that a subset of genes involved in tumorigenesis and immune responses displayed increased dysregulation in EnKS lesions, but the overall gene expression profiles between EnKS and EpKS correlated strongly. Investigation of cellular transcriptomes through RNA-seq have also revealed important aspects of nuclear DNA sensor regulation outside of the context of virus infection. To provide a few examples, expression of IFI16, among several other innate immunity proteins, was upregulated in macrophages infected with the bacterium Campylobacter concisus [102]; tumor-bearing mice with deletion of the IFI16 homolog p204, when compared to WT mice, lacked the ability to induce the upregulation of 382 genes, indicating the extensive involvement of IFI16 in antitumor immunity [57]; and RNA-seq studies of an alcohol-related liver disease model in mice revealed that liver damage from excessive alcohol consumption is mediated by cGAS activation of the STING–TBK1–IRF3 pathway [103]. Similar to transcriptome studies, whole-cell proteome investigations with mass spectrometry have led to a wealth of information about both viral and cellular protein abundances during virus infection, uncovering changes linked to innate immune responses and virus immune evasion strategies. Given the finely tuned temporal regulation of virus replication steps, assessments of the cellular proteomes have been carried out at multiple time points as the infection progresses, as reported for infection with HSV-1 [51,104], HCMV [105,106], and KSHV [107,108]. In conjunction with temporal studies, infection with virus strains that lack the ability to inhibit DNA sensors offered a view of proteome changes during an active host immune response. For example, the d106 HSV-1 strain contains mutations in four of five immediate-early proteins (ICP4, ICP22, ICP27, and ICP47) but expresses functional ICP0 [109]. Infection with this virus results in increased induction of cytokines and apoptosis when compared to infection with WT HSV-1 [50,110]. By comparing temporal proteome changes during WT and d106 HSV-1 infections, we discovered the upregulation of several proteins involved in innate immunity and apoptosis, and integration with cGAS IP-MS led to the discovery of OASL-mediated cGAS inhibition [51]. Additional MS studies have been carried out to characterize proteome changes during HSV-1 infection in a range of cell types and to compare alterations induced by different virus strains [51,54,104,111–121]. Spatial proteomics [122] has further provided the ability to characterize changes in proteome organization during infection [123], as well as discover viral proteins that localize to distinct organelles to regulate their functions, as shown for HCMV infection [124]. Recent years have also seen the increased integration of proteome studies with global PTM studies, where the infection-induced host phosphorylation, acetylation, SUMOylation, ubiquitination landscapes, to name just a few, have been started to be characterized [125–128]. Knowledge of global PTM changes have furthered the understanding of signaling cascades during infection and have helped to identify regulatory hubs at the interface between host defense and virus production. Another proteomic perspective of regulatory hubs is provided by the identification of functional protein complexes that are activated or inhibited during an infection process. The use of thermal co-aggregation profiling MS was recently demonstrated to offer a global view of temporal assembly and disassembly of host–host, host–viral, and viral–viral protein interaction events during HCMV infection, including the regulation of complexes involved in host immunity [106]. Altogether, these MS-based proteomic investigations of whole-cell and subcellular proteomes, interactomes, and PTMs provide rich information regarding host cell changes in response to viral infections. The integration of these different datasets promises to reveal a systems-view of the host environment during infection, which can aid in the formulation of specific biological hypotheses, the identification of changes linked to viral pathologies, and the discovery of therapeutic targets. Therefore, efforts have been and continue to be placed in the development of computational platforms that facilitate data integration in a user-friendly manner [129–135]. One platform specifically applied to studying viral infections is the Interaction Visualization in Space and Time Analysis (Inter-ViSTA), a web-accessible platform that enables integration of interactome, proteome, and functional traits to build animated temporal interaction networks [136]. For example, this analysis platform readily illustrated dynamic localization-dependent interactions of the HCMV protein pUL37 that function to either inhibit immune responses early in infection or promote peroxisome metabolic functions that benefit virus assembly late in infection. Metabolome profiling brings another powerful omic tool to understanding the biology of virus infection and host defense mechanisms. Replication and assembly of virions is an energy-intensive process that requires the virus to trigger the cellular machinery to increase protein and lipid production for building progeny virions, as shown for numerous viruses [137]. Great effort has been put into understanding the mechanisms underlying metabolic reprogramming during a number of viral infections, including with HCMV and HSV-1 [55,138]. Integrating MS-based metabolomics with molecular virology techniques has proved valuable towards this goal; for example, a recent study of HCMV infection found that the viral protein pUL37 is critical for remodeling cellular metabolism by increasing production of very-long-chain fatty acids [139]. Given that pUL37 is an important immune evasion protein, such as by inhibiting cGAS function [39], it is likely that pUL37 bridges proviral metabolism with innate immune regulation during HCMV infection. Future studies geared towards elucidating the relationships between these fundamental infection processes promise to reveal key players in virus replication and spread. Finally, the secretion of proteins into the extracellular space is crucial for communication with adjacent cells and is the foundation of innate immunity. Interferons secreted by infected cells bind to receptors on neighboring cells to induce immunomodulatory and antiproliferative effects, a phenomenon that has been known for several decades [140]. Upon binding to the interferon receptor and activating the JAK–STAT signaling pathway, dozens of transcripts are upregulated, including additional cytokines [141], altogether leading to inflammatory response and impacting disease pathology. Therefore, examining the secretome of infected cells is a necessary component for understanding these complex intercellular communications [142]. MS-based studies have leveraged proteomics and lipidomics methods to define the composition of secreted biomolecular complexes during infection, including extracellular vesicles known as exosomes [143]. For example, quantitative proteomic analysis of exosomes from HSV-1-infected macrophages demonstrated that specific subsets of cytokines, inflammatory proteins, and transcription factors are secreted rapidly upon infection, thus priming immune response in neighboring cells [144]. Virus-driven secretomes can also impact cellular and tissue physiology, as demonstrated by two recent studies that examined how molecules secreted by herpesvirus infected cells determine local immune and growth responses in neutrophils [145] and cortical brain cells [54], respectively. #### 4. The Missing Link: Genomics for Understanding the Viral DNA–DNA Sensor Interface AP-MS isolations of viral DNA during infection have been fundamental for the discovery of nuclear DNA sensors. However, the regulation and complete outcome of the interactions between DNA sensors and viral DNA remain to be fully characterized. In this section, we discuss the conundrum of how DNA sensors bind to pathogenic DNA in a sequence-independent manner, while also being shown to specifically function in repression of viral gene expression. Though nuclear DNA sensors avoid autoreactivity with host DNA, they do not appear to recognize any specific virus nucleotide sequence motifs or DNA modifications. In fact, for a protein to be classified as a DNA sensor, one requirement is that it should bind to DNA in a sequence-independent manner, thereby having the capacity to recognize multiple DNA pathogens. For example, for the HIN-200 domains of IFI16 and IFIX, their sequence-independent binding to dsDNA is accomplished via weak electrostatic interactions between positively charged amino acids and the negatively charged DNA phosphate backbone [25,146,147]. It was also demonstrated that IFI16 preferentially binds to specific DNA forms, namely cruciform structures, superhelical, and quadruplex DNA, which could maximize contact between the phosphate backbone and the basic amino acids in the HIN-200 oligonucleotide/oligosaccharide binding folds [148,149]. However, there remains no evidence of DNA sequence preference, and it is hypothesized that the activation of immune responses by IFI16 relies on cooperative assembly of IFI16 oligomers, which is limited on host DNA by tight chromatin packing [29,150]. Examinations of crystal structures of cGAS with a dsDNA ligand have similarly shown that the cGAS Mab21 domain binds to the phosphate backbone of B-form DNA without any sequence specificity [151–154]. In contrast with IFI16, it is proposed that cGAS-mediated autoreactivity is inhibited by tight tethering of cGAS to host chromatin through a salt-resistant interaction that is independent of the domains required for cGAS activation [34,35]. Such in vitro experiments indicate that DNA binding is sequence independent, but the propensity of DNA sensors to interact with transcriptional regulatory proteins that are sequence specific (e.g., the HSV-1 transcriptional activator ICP4 [155]) could induce preferential accumulation at certain DNA loci. Furthermore, given that IFI16 and IFIX have also been shown to function in host antiviral response by repressing virus transcription [29–33], how does DNA sensor binding affect the chromatin structure at specific binding sites? Are other protein–DNA interactions increased or decreased at these loci, and how does this affect viral transcription and replication? After entering the nucleus, herpesvirus genomes are subjected to chromatinization by host cell histones [156], and it has been demonstrated that IFI16 promotes the addition of the repressive heterochromatin mark H3K9me3 on viral DNA [31,32,157]. Thus far, these studies investigating where IFI16 and H3K9me3 interact with viral genomes have been conducted using chromatin immunoaffinity purification (ChIP) coupled with PCR or RT-qPCR [31,32,157]. Herpesviruses have large genomes (e.g., HSV-1 is ~152 kilobase pairs and contains ~80 genes), yet this approach is limited by only examining protein–DNA interactions at a few viral genes. Higher throughput techniques can help to more broadly represent interactions between viral DNA and DNA sensors and the subsequent effects on the viral genome chromatin landscape. To assess where DNA sensors bind to the viral genome, ChIP sequencing (ChIP-seq) is an appropriate technique that has previously been used to study how the HSV-1 genome interacts with ICP4 [155], RNA polymerase II [158], and the transcription factor CCCTC-binding factor (CTCF) [159]. Applying this technique with nuclear DNA sensors would help determine whether DNA sensing is fully a sequence-independent process or whether additional factors within the cell can also cause accumulation of the DNA sensor at specific DNA loci. Histone PTMs such as H3K9me3 are often used as proxies for determining whether a DNA locus resides in a euchromatin or heterochromatin region of DNA [160]. To investigate how DNA sensors affect the chromatinization of viral genomes, knockout studies can be followed by H3, H3K4me3, and H3K9me3 ChIP-seq. However, these modifications only act as a proxy for the chromatin structure and are not a direct readout of chromatin structure. Additionally, the cost of such experiments must also be considered, as the requirement for multiple conditions per sample considerably increases the amount of sequencing required. Measuring chromatin accessibility is often a better way to examine chromatin structure and can be probed through techniques such as MNase-seq [161], DNase-seq [162], FAIRE-seq [163], and ATAC-seq [164]. Furthermore, integration of protein–DNA interaction mapping data with chromatin accessibility data following DNA sensor knockout can help to identify how DNA sensor binding both globally and locally affects viral DNA structure. Thus, high-throughput sequencing techniques that explore epigenomic changes will be pivotal to continuing to expand our understanding of nuclear DNA sensor mechanisms. #### 5. Concluding Remarks The development of omics techniques has helped to greatly expedite biological research. The topic discussed in this paper, the elegantly complex process of nuclear DNA sensing during virus infection has benefited immensely from the ability to examine the identities and PTM states of all proteins within the host cell. The general idea behind DNA sensors is rather simple: bind pathogenic DNA and initiate antiviral signaling pathways. However, the mechanisms by which the nuclear DNA sensors IFI16, IFIX, cGAS, and hnRNPA2B1 activate large-scale transcriptome, proteome, and secretome changes rely on the precise coordination of a multitude protein interactions and PTMs. Here, we have discussed how omics techniques, particularly those implementing mass spectrometry, have led to the discovery and characterization of these nuclear DNA sensors. The future expansion of these investigations to integrative multiomics studies that include epigenomic assays promise to substantially contribute to a more in-depth understanding of the intricacies of DNA sensing, its dysregulation, and connected pathologies. Author Contributions: Conceptualization, T.R.H. and I.M.C.; writing—original draft preparation, T.R.H. and I.M.C.; writing—review and editing, T.R.H. and I.M.C. All authors have read and agreed to the published version of the manuscript. Funding: We are grateful for funding from the NIH (R01 GM114141) and Mallinckrodt Scholar Award to IMC, and from the NIH training grant from NIGMS (T32GM007388). Conflicts of Interest: The authors declare no conflict of interest. #### References Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com Biomolecules Editorial Office E-mail: [email protected] www.mdpi.com/journal/biomolecules MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel: +41 61 683 77 34 Fax: +41 61 302 89 18 www.mdpi.com ISBN 978-3-0365-2583-9	doab	2025-04-07T03:56:58.982438	11-1-2022 14:51	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0067e0a8-b387-4d2e-a700-d437204e9c1c", "url": "https://mdpi.com/books/pdfview/book/4705", "author": "", "title": "Integrative Multi-Omics in Biomedical Research", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036525822", "section_idx": 2 }
006d7857-e96f-4630-aa40-deadb120d9aa	## SPRINGER BRIEFS IN HUMANCOMPUTER INTERACTION # Jacob D. Oury Frank E. Ritter Building Better Interfaces for Remote Autonomous Systems An Introduction for Systems Engineers ## Human–Computer Interaction Series SpringerBriefs in Human-Computer Interaction ## Editors-in-Chief Desney Tan Microsoft Research, Redmond, WA, USA Jean Vanderdonckt Louvain School of Management, Université catholique de Louvain, Louvain-La-Neuve, Belgium SpringerBriefs in Human-Computer Interaction presents concise research within the fast growing, multidisciplinary feld of Human-Computer Interaction (HCI). Designed to complement Springer's prestigious Human-Computer Interaction Series, this Briefs series provides researchers with a forum to publish cutting-edge scientifc material relating to any emerging HCI research that is not yet mature enough for a volume in the Human-Computer Interaction Series, but which has evolved beyond the level of a journal or workshop paper. SpringerBriefs in Human-Computer Interaction are shorter works of 50–125 pages in length, allowing researchers to present focused case studies, summaries and introductions to state-of-the-art research. They are subject to the same rigorous reviewing processes applied to the Human-Computer Interaction Series but offer exceptionally fast publication. Topics covered may include but are not restricted to: SpringerBriefs are published as part of Springer's eBook collection, with millions of users worldwide and are available for individual print and electronic purchase. Briefs are characterized by fast, global electronic distribution, standard publishing contracts, easy-to-use manuscript preparation and formatting guidelines and have expedited production schedules to help aid researchers disseminate their research as quickly and effciently as possible. More information about this subseries at http://www.springer.com/series/15580 Jacob D. Oury • Frank E. Ritter # Building Better Interfaces for Remote Autonomous Systems An Introduction for Systems Engineers Jacob D. Oury Applied Cognitive Science Lab, College of Information Sciences & Technology Pennsylvania State University University Park, PA, USA Frank E. Ritter Applied Cognitive Science Lab, College of Information Sciences & Technology Pennsylvania State University University Park, PA, USA ISSN 1571-5035 ISSN 2524-4477 (electronic) Human–Computer Interaction Series ISSN 2520-1670 ISSN 2520-1689 (electronic) SpringerBriefs in Human-Computer Interaction ISBN 978-3-030-47774-5 ISBN 978-3-030-47775-2 (eBook) https://doi.org/10.1007/978-3-030-47775-2 © The Author(s) 2021 . This book is an open access publication. Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifc statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland To my parents, Molly and John Oury, for always putting up with my antics and keeping my head from getting too big. (Oury) To my mentors and mentees who have done the same for me. (Ritter) ## Preface This brief book Building Better Interfaces for Remote Autonomous Systems: An Introduction for Systems Engineers, which we shorten to Building Better Interfaces here, originated from work that we have done with L3Harris Technologies (formerly Harris Corp) on improving interface design for operations centers. We realized that this work could be valuable to a wide range of designers and engineers, especially in felds that have typically not prioritized interface design in their projects. We wrote this book for the engineers, designers, and managers that are responsible for building large, multi-team systems found in places like NASA's control rooms or control rooms for nuclear power plants. This book gives specialized engineers and developers a broad review of important design frameworks and knowledge about how operators see, think, and act so they can make better decisions and better interfaces. It is a brief book for busy designers to quickly introduce these issues and some of the many ways to improve interfaces. Thus, it is part of the SpringerBriefs in Human-Computer Interaction. In the past several years, the signifcance of interface design has become more apparent; specialized user experience design teams are becoming more common in unexpected places like the defense industry. As recognizing the importance of usability becomes more common, we hope that this book can help shape the discourse regarding how interface design fts alongside more well-established felds like electrical engineering. This book advocates for user-centered design, rather than user experience design, as the central goal of the team handling interface design. User experience caters to the user, focusing on how they feel or respond emotionally to design choices. This is a less useful and less appropriate approach for the types of systems we discuss in this book. It can be very appropriate for consumer products. In contrast, user-centered design takes the user off a pedestal and places them onto equal footing with the rest of the system as simply another subsystem or component. This makes stakeholders and designers assess risks to project failure more accurately for systems that require human input. Failure of any subsystem, even the human operator, can lead to disaster. Every component has safe operating conditions that give reliable results; this book demonstrates how you can begin applying those same standards to the operator and their interactions with other systems. This book is suitable for undergraduates studying any feld and system designers. It is designed to be a standalone document. Readers with some experience in interface design and psychology may fnd some sections trivial, but we hope that every reader will gain some value from having read it. For those wanting a deeper review of these topics after fnishing this book, we recommend Foundations for designing user-centered systems by Ritter, Baxter, and Churchill. In many ways, Building Better Interfaces is a practical application of the lessons from Foundations for designing user-centered systems for designing remote, autonomous systems. College of IST Jacob D. Oury The Pennsylvania State University University Park, PA, USA Frank E. Ritter ## Acknowledgments An early draft of this book was produced as part of a project with L3Harris Technologies. This project and book wouldn't have come together without the support of Mark Wynkoop, Tom Wells, Jim Ringrose, Gisela Susanne Bahr, and the other current and former members of the Specialty Engineering UX Team including Alison Sukolsky, John Blood, Craig Pickering, and Hanna Clark. Finally, Mark Foster provided incredible insight and was the primary designer of the Water Detection System used as an example in this book. We greatly appreciate the engaging discussions and comments on this book from our colleagues, friends, and mentors associated with the Applied Cognitive Science Lab at Penn State including Sarah Stager, David Reitter, Shan Wang, Raphael Rodriguez, Pooyan Doozandeh, Chad (Chungil) Chae, April (Yu) Yan, Farnaz Tehranchi, and Caesar Colchado. We also appreciate the extensive and very useful comments from our colleagues Steve Croker and Gordan Baxter. Sven Bilen gave sage advice on key occasions. We also thank Helen Desmond, who was patient and helpful during the development of this book, and an anonymous Springer copyeditor helped in preparing and publishing this book. The opinions are those of the authors and do not necessarily represent those of L3Harris. ## Contents ## Chapter 1 Introducing Interface Design for Remote Autonomous Systems Abstract This chapter presents a high-level overview of how designers of complex systems can address risks to project success associated with operator performance and user-centered design. Operation Centers for remote, autonomous systems rely on an interconnected process involving complex technological systems and human operators. Designers should account for issues at possible points of failure, including the human operators themselves. Compared to other system components, human operators can be error-prone and require different knowledge to design for than engineering components. Operators also typically exhibit a wider range of performance than other system components. We propose the Risk-Driven Incremental Commitment Model as the best guide to decision-making when designing interfaces for high-stakes systems. Designers working with relevant stakeholders must assess where to allocate scarce resources during system development. By knowing the technology, users, and tasks for the proposed system, the designers can make informed decisions to reduce the risk of system failure. This chapter introduces key concepts for informed decision-making when designing operation center systems, presents an example system to ground the material, and provides several broadly applicable design guidelines that support the development of user-centered systems in operation centers. ## 1.1 Introduction Our increasingly complex society relies on an interconnected network of systems, each responsible for carrying out its own role effectively. The most important components within the systems of systems are called critical systems. Critical systems are defned by the cost of their failure; critical systems are called as such because their failure will lead to loss of life, destruction of the system, or failure for the organization as a whole. For example, failure in central command for the space missions may leave astronauts without the information (and oxygen!) they need if their oxygen tank were to fail a few days into the mission. Air traffc control is another example of a critical system; even minor mistakes can have devastating consequences. Not every critical system, however, needs to be part of a large international organization. A 911 emergency call center is responsible for triaging calls, dispatching appropriate services, and providing support for the caller; loss of the call center means local fre, medical, and police services lose their ability to coordinate and respond. Whether it's NASA's Christopher C. Kraft Jr. Mission Control Center in Houston, the Indianapolis Air Route Traffc Control Center, or a local 911 dispatcher, these critical systems all contain some form of an operation center at the heart of their operation, and these operation centers are vital communication hubs for the transfer of information. Within any given op center, there are going to be different stakeholders, tasks, and priorities that must be considered in their design. A single room or even a single screen could be the link between the op center and multiple complex systems. Figure 1.1 shows a montage of the types of system components this book addresses. This book primarily examines operation centers that manage remote, autonomous, asynchronous systems. The book is designed to be useful to managers, designers, and implementors of op centers. Managers can use it to adjust their process to account for a wider range of risks caused by failing to support their users and their tasks. Designers can use it to manage the process, learn about users, and become more aware of useful types of shared representations. Implementers can use it to provide context for seemingly small decisions within an interface that are too minor to be described formally or have not been specifed. Where we can, we also identify design principles and aspects of the operator, interface, or process that suggest prescriptive actions to create better interfaces. This introductory chapter makes the case for including knowledge about users as part of the system and design process. It will then briefy describe a way to include this knowledge (the Risk-Driven Spiral Model) and how this knowledge could be applied to operation centers. The rest of the book will use an example system called the Water Detection System (WDS) to help illustrate the principles, concepts, and practical implications derived from the material covered. The introduction concludes with some example guidance that can be used as an executive summary or as a summary for readers who might not have time to read the whole book. The remainder of the book provides support for the guidelines. The appendices include a worked example that shows how the guidance is applied. Table 1.1 defnes some common terms used throughout this book. The design approach that results from this book will be primarily a human–computer interaction (HCI) approach to make the system usable. Aspects of improving the system through user-centered design (UCD) and making the system more enjoyable (while maintaining usability) with user experience (UX) design will be included as well. ## 1.2 The Role of Operators Operators can greatly infuence operation center success. In a study of errors in air traffc control, a type of op center, Jones and Endsley (1996) found that seven out of ten times system failures are due to operator error. Their error analysis for Fig. 1.1 Technological advancement has expanded our ability to use and control complex systems in new ways and from new locations. To make full use of these powerful new systems, usability is paramount. (Image by Kenan Zekić) Table 1.1 Common terms and defnitions aviation disasters organized the contributing errors by operators using Endsley's (1995) theory of situation awareness. The situation awareness framework predicts operator performance by rating the operator's awareness of necessary information. When the errors were organized into their stage of situational awareness, they found that misperception or non-perception of the necessary information was the primary cause of air disasters about 75% of the time. Going up in complexity, failing to successfully comprehend the meaning or the importance of information was the primary cause in only about 20% of air disasters. Finally, at the lowest error rate, projection into near-future system states is the key in less than 5% of disasters. Breaking down these failures into more specifc types of failure showed that attentional failure (35%; operator has information but fails to attend to it), working memory failure (8.4%; operator attends to information but forgets it), and mental model failure (18%; operator's understanding of the situation does not match reality) account for the most common events that contribute to operator errors in op centers. Operators of complex systems use a set of cognitive mechanisms that are fallible in predictable ways. Systems engineers, developers, and designers can begin mitigating the risks associated with fallible cognitive behavior by learning about the factors and mechanisms that infuence operator performance and reliability. Not all these mechanisms can be ameliorated by system design, but they do shed light on design opportunities where systems could be improved and better support operators. This book suggests ways to do that. Modifying op center designs could help reduce these types of system failures by providing the information more clearly, making information more comprehensible, requiring less attention (perhaps by reducing other less useful information), and appropriately matching and supporting the operator's mental model and tasks. How can these issues be addressed throughout the development cycle of complex systems? We propose a design process based on understanding the operator, their tasks, and the technology. ## 1.3 How to Improve Designs The variety and complexity of work being performed in op centers prevents strict design guidelines from being a "silver bullet" for every system design issue. The different goals, priorities, and tasks across op centers will likely add up to being nearly equal to the number of op centers itself. However, the common element across op centers is the role of human operators. Operators serve as the interface between the wide range of information sources and the higher command structure. This can involve a vast variety of tasks ranging from call intake and prioritization within an emergency response center to monitoring radar for airborne threats. Furthermore, the task variety is compounded by having a single operator be responsible for multiple tasks. For example, an operator at a 911 dispatch center will often be simultaneously responsible for (a) providing emotional support and guidance to the caller, (b) recording crucial information about the situation, (c) alerting appropriate emergency responders, and (d) answering questions for emergency responders while en route. The complexity and variety of tasks within an op center means that the system designers will need to know their users, their users' tasks, and the technology and then combine these using their judgment within the design process. At all times, designers must be aware that interfaces that are hard to read, use, understand, or predict from are constant risks to project success; however these issues are not always easily solvable. Designers will have to use judgment when aspects of the users and their tasks are not fully known. They will also have to use judgment to prioritize tasks or user types and to balance different design requirements. Designers face many challenges when balancing human and system factors, and this book will help guide their decision-making when solutions are not immediately clear. Simply providing a set of design guidelines will not suffce, because one size does not ft all. Due to the varied nature of tasks and systems across operation centers, we will need to provide a suitable foundation for designers to guide their decision-making when there is no direct solution. Thus, this book summarizes a useful process and design issues to keep in mind when designing operation centers. It goes further, however, by providing a worked example of design and design steps for an example system. This book spends more time defning a useful interface design process than giving simple guidelines for design. This user-and-task-oriented process should lead to better interfaces that support operators and do this in a better way than simply providing a set of ten "rules" about font size, which might need to vary and which will confict at times with rules about how many objects need to be visible on the interface. And, yet, in providing background knowledge about operators and their tasks, there will inevitably be sensible conclusions that look like and work like guidelines. The design recommendations will often provide "safe" recommendations for designers. Design recommendations will be accompanied by brief supporting details meant to substantiate the information. This self-contained book will provide system designers with a framework for improving user experience and performance by incorporating human-centered design principles into the design and implementation of critical systems. System designers will beneft greatly from understanding the foundational concepts and literature that support this guidance. This book provides a simple review of the literature to support this guidance. This review serves several purposes: (a) offering motivation for including the topics chosen, (b) describing the related research that has contributed to the high-level guidance, and (c) providing readers with a convenient method to learn more about a topic if needed. While not every system developer will choose to read this book, it provides interested readers with a more condensed treatment than available from reading several books on user-centered design and users. The fnal review and guidance should be detailed enough to provide further guidance in a standalone format. ## 1.4 Risk-Driven Design The design and performance of an operation center will depend on fnancial considerations, task constraints, and the goals of the designers. However, clearly there are limitations on what is possible for any given design process (e.g., deadlines, access to user testing, ambiguous information). In an ideal world, every project would have ample time, personnel, and funding to be able to create the best product possible: clearly this is an unrealistic scenario. Thus, designers and other stakeholders must make decisions about how to ensure project success throughout the design process. We propose that the Risk-Driven Incremental Commitment Model (RD-ICM) provides the best framework for creating effective systems, including assessing the risks associated with design choices (Pew and Mavor 2007). Figure 1.2 shows the RD-ICM in spiral form. Implementation of RD-ICM involves assessing the risk associated with a given decision. Boehm and Hansen (2001) defne risks within the RD-ICM as "situations or possible events that can cause a project to fail." RD-ICM uses an iterative, fexible procedure to prompt the stakeholders to make candid assessments of what the risks are at each stage of the project. Implementing RD-ICM effectively leads to decisions contrary to the dogmatic idea that UX be prioritized at every stage, but this is because UX issues are only explored once their risks are relatively large. The RD-ICM and risk-driven design require four key features: Fig. 1.2 The Risk-Driven Incremental Commitment Model as a spiral of development. (Reprinted from Pew and Mavor 2007, p. 48) 4. The process explicitly takes account of risks during system development and deployment to determine prioritization for resource deployment: minimal effort for minimal-risk decisions, high effort for high-risk decisions. Within the spiral, each stage has phases of (a) stakeholder valuation and evaluation; (b) determination of objectives, alternatives, and constraints; (c) evaluation of alternatives and identifcation and resolution of risks; and (d) development and verifcation of the next-level product. This approach allows work on risks to proceed in parallel and comes back to value the alternatives with the stakeholders. Here is an example of how the RD-ICM could shape design choices. During the early design process of a complex system, the risks of not getting the system up and running (e.g., failure to meet expectations for funders or other high-level stakeholders or technical connection issues) may outweigh the risks associated with having a nonideal interface design (e.g., frustrated users). The stakeholders have determined that functionality (the task-related aspects of the design) should be prioritized over the user experience (UX, the users' feelings, emotions, values, and responses to the system). Instead, the UX design choices could be pushed down the pipeline and then reassessed at a later stage. This would enable the engineering team to focus on creating something that "works." However, once a functional system is formed, the team would reassess the risks associated with a frustrating user interface. If the interface fails to convey critical information in a consistent manner to most users, the risks of a user misinterpreting a signal may outweigh the benefts of adding further features to the system. Each stage has its own iterative assessments of how to successfully complete the project. Further information on this approach is available from a National Research Council Report (Pew and Mavor 2007), a special issue of the Journal of Cognitive Engineering and Decision Making (Pew 2008), and an overview in the Foundations for Designing User-Centered Systems textbook (Ritter et al. 2014). So, if you adopt a risk-driven process that includes human operator-related risks, you still must be able to recognize and reduce these risks. This book seeks to provide background knowledge to help developers judge and ameliorate the risks to system success that developers face during the design and implementation process of op centers. We hope to provide knowledge and guidance that can help designers understand how their design choices may affect task performance throughout the lifetime of the system. Thus, we suggest following a risk-driven spiral model. This includes formal reviews with stakeholders at each cycle to assess risks and work focused to reduce risks, not just build a system. This approach uses a range of design documents as shared representations between the stakeholders and the designers and implementers. We include an example set in Appendix 1. ## 1.5 The Design Problem Space for Op Centers This book reviews how the risks of failures due to human performance can be alleviated throughout the design process of interfaces within operation centers. Because designing an interface for an op center is the design problem, we briefy review this design space and provide an overview of an example before addressing further common risks and issues that apply to operator interactions with the systems. Op centers act as the nervous system within a larger body, directed to monitor or respond to a set of events. The op center aggregates information input and output to facilitate a rapid response to changing conditions. The specifc procedures used are typically guided by senior staff, while operators themselves will be responsible for interpreting information, transmitting orders, and following preset procedures for specifc situations. There are three components to this design problem: the technology to support and implement the system, the users, and the users' tasks. The frst item is briefy noted as an important component that will support and constrain designs. The fnal two are the focus of this book, so we address them together. ## 1.5.1 Know Your Technology Across the range of stakeholders involved with the design of a system, the most infuential stakeholders will likely prioritize system functionality over concerns of operator-related risks like improving user-centered design. While this may irk the designers of human-facing subsystems, this basic fact should infuence how the design process is conducted. Thus, system designers should have at least some understanding of how the technology within their system functions. The underlying, unmanned technology within op centers processes and transmits the information that is presented to an operator. So, the frst issue in design is to know what the technology can and cannot do. The technology in an op center is likely built from varied inputs and outputs, ranging from manually entered paper documentation to antenna arrays linked to distant sensors. On its own, a component like an oxygen sensor simply outputs an associated metric. However, once integrated into an environmental monitoring station in an op center, additional design features to support human use (i.e., an interface, optional controls, and memory for time series) become apparent. Interface designers may not need to understand the intricacies of each component but should have some knowledge of the technology associated with their system. The types of systems built for op centers are likely to differ greatly in their underlying technology and purpose. In some cases, designers can grasp the underlying technology well enough to create effective systems, but this may not always be the case. Building an electrical circuit monitoring system and building a hydrothermal monitoring system may require incorporating subject matter experts into the design process, especially for high-stakes systems like a nuclear power plant. Finally, designers should understand the tools they need to build interfaces as well. The interface tools need to be able to support the designers in creating usable interfaces, which not all tools support well (Pew and Mavor 2007; Ritter et al. 2014). To our previous example, an electrical circuit monitoring system may require designers to reference an unfamiliar program used by electrical engineers like Pspice (Personal Simulation Program with Integrated Circuit Emphasis). Stakeholders should ensure that system designers can successfully understand and utilize the necessary information. Understanding the technology within the system and used to build the system will help with the inevitable design choices. The typical issue is where designers should ft the person to the machine vs. ft the machine to the person. Sometimes, technological or personal constraints will prevent designers from optimizing the ft in one direction or another, but knowing the technology will help reduce problems of ft in both directions. ## 1.5.2 Know Your Users and Their Tasks On the other hand, designs that do not support users to do their tasks can fail for this reason as well, so system designers need to study the user and how to design for users. The focus of this book is to explain how to know the users of the op centers, the operators, and their tasks. Human operators and their tasks, in many cases, will be as complex as the technology. The only difference is that many technology designers have been trained in technology design, but not in the science of how operators think, learn, and do their tasks. This book notes some of the literature, results, and methods for understanding operators to help in the design process. Similarly, it describes methods for improving the work process, like task analysis (TA), which is a useful tool for specifying, implementing, and checking op center designs. The technology may be able to deliver, but will the operator be able to understand and use the system at the expected speeds? Will the tasks, including their microstructure and dependencies, be supported? Or will the operator have to correct and store information (in a more fragile memory than computer memory)? These types of mismatches between operator and system are frequent causes of system failure. The gold standard in design (Card et al. 1983; Pew and Mavor 2007; Ritter et al. 2014) is to know the operators, know what tasks they are trying to perform, and then use the technology as best as it can be used, to support the tasks based on the operator's capabilities. Designers who use their own understanding of a system as a reference (instead of that of the actual users) commit the fundamental attribution error and risk-creating systems that are unwieldy or outright unusable by the intended users (Baxter et al. 2014). The fundamental attribution error of design refers to when designers assume all users are just like themselves. As we note in our example system in this book, this is often a mistake and leads to problems in usability because the designer and the operator have different knowledge, skills, and abilities. In addition, leaving out tasks or making them less easy to perform, or making state information visible only upon query, are all mistakes that are easily avoided, but require knowing the operators and their tasks. Knowing the frequency and importance of tasks is also important. Common and important tasks should be more easily and safely accomplished than less common and less important tasks. When the two factors of frequency and importance collide, then possible design choices become apparent. At this point designers can assess the situation through the RD-ICM and reduce risk by getting feedback from stakeholders, researching similar design problems, or testing multiple designs depending on the risks associated with each choice. There are numerous guidelines on how to create task analyses (e.g., Cox 2007; Ritter et al. 2014, Ch. 11). There are tools to support TA (i.e., Cogulator1 ), but often plain text documents provide the best value and are useful enough for most designs. <sup>1</sup> http://cogulator.io/ TA is a lot like pizza—while the balance of contents may vary in approaches, most versions are usable and enjoyable. ## 1.5.3 Test Designs Broadly and with Cognitive Walkthroughs During design and implementation, there may be unknown aspects of the users, their tasks, or the interactions between the two. A way to reduce the risk of system failure is to test the resulting system. The test can be quite simple, for example, simply to see if the tasks can be performed. Alternatively, there are more complex methods, like running a small A/B experiment with two possible designs or measuring task performance with actual users under realistic conditions. Pew and Mavor (2007) review the range of these tests, and there are multiple textbooks describing them (e.g., Cairns and Cox 2008; Lewis and Rieman 1994). Testing interfaces will reduce the range of usability risks, but test methods vary by how much of a time and resource commitment is required to get useful results. Asking someone unfamiliar with the project to review the proposed interface mockup may be essentially free, whereas conducting an A/B test with expected users may take weeks (if not months) to fully set up, run, and analyze, but will be much more useful. The simplest test is to have naïve operators use the interface and observe them. This approach is explained in many textbooks, including Ritter et al. (2014). Such tests with naïve users could last as little as 10 min and cost next to nothing (i.e., ask a colleague to use the interface and provide comments) or could take multiple months and cost \$100 k (i.e., conducting a formal study on task performance under realistic conditions). Stakeholders should consider system requirements and risks to determine how their system should be tested. We also support using "cognitive walkthroughs" (Polson et al. 1992) to examine the usability of the system. A cognitive walkthrough is a method for evaluating the learnability and usability of an interface by simulating the cognitive activities of a typical user during normal tasks. The typical process for performing cognitive walkthroughs begins with describing the goals and tasks that are required by the system. First, the goal structure of the model is generated from expert interviews, prior research, and other forms of information gathering. The goal structure, like a task analysis, is arranged into a hierarchy. The top-level goals represent the overall task. Each top-level goal is composed of intermediate-level goals (subtasks), each of which is composed of a set of individual actions. Cognitive walkthroughs, when performed successfully, should determine whether the operator of a system is making the correct connections between each level of the goal. That is, the analyst compares the goals with the interface and attempts to map how a typical user would accomplish each goal, subtask, and action. If the analyst cannot make some mapping of a goal to the interface, this will suggest an area of the interface that requires improvement or further work. One potential pitfall here can occur if the analyst is too familiar with the interface (relative to a true "typical user"), as they will not see the same problems that users will see, at least novice users. The data collected from cognitive walkthroughs can enable developers to provide supplementary "clues" or signals to the operator at specifc locations to ensure that each goal, sub-goal, and individual action provide a coherent information set capable of being understood and followed by the operator (Blackmon et al. 2002; Polson et al. 1992). Cognitive walkthroughs require a task analysis and thus will take between an hour and a short working day to perform in most cases. The length of time is based on the number of tasks and how diffcult they are to perform. Cognitive walkthroughs may require domain knowledge and thus may be performed in teams comprised of an analyst working through the task analysis and a domain expert making the decisions. Whenever detailed time predictions are useful, we recommend using the keystroke-level model (KLM) of Card et al. (1980, 1983). This approach provides time estimates based on the keystrokes, mouse moves, mental operators, system response time, and other possible cognitive operators. The times are engineering estimates (i.e., ± 20%), but basically support fair comparisons of different interfaces. The KLM time predictions suggest where and how time is spent on an interface and can help identify ways to improve performance. The regularity of the interactions across subtasks also suggests how much needs to be learned by the users and where knowledge may be misapplied. There are numerous ways to reduce system failure due to usability problems. This section noted a few and how to fnd more. Next, an example system is introduced to ground this discussion and show examples of how potentially abstract principles can be put into practice. ## 1.6 Example Task: The Mars Water Detection System This book provides context for readers through a hypothetical use case for a semiautonomous system that searches for water. The scenario is based on designing an op center for command and control of a remote Water Detection System (WDS) to accompany a manned mission to Mars. The WDS is a mostly autonomous mobile robot that searches Mars for signs of water, but the WDS sometimes requires human intervention to respond to novel or risky scenarios. The WDS will arrive alongside the mission team and begin operation following its assembly by the team. Following its activation and an initial system check, the op center on Earth will take over sole command of the WDS for a 10-year mission. Scientists in the program offce will make high-level decisions to support the mission of fnding water, while the Earth-based operators implement action plans and monitor the various systems for any current or upcoming issues. The rest of this chapter provides a brief review of the WDS and its design requirements before concluding with some design recommendations that arise from this chapter. A detailed description is presented in Appendix 1. ## 1.6.1 Operation Center Organization The WDS is one part within the larger structure of an op center hosting dozens of systems that require constant oversight. While the WDS is important for the mission, it may not be the primary focus for the workers at any given time. The command structure of the op center involves bidirectional communication between scientists from the Program Offce who funded the WDS and the operators responsible for direct interaction with the systems. Figure 1.3 shows a few example interface prototypes for the WDS. While the design will vary depending on the needs of the system, these systems present many different metrics of system performance. Operators will monitor the system, pass along alerts, and update the alerts depending on their risk assessment for a given situation. Scientists will take this information and pass back commands for the operators to transmit. Certain tasks will be Fig. 1.3 Two example interface designs for the Water Detection System monitoring screen (second example on next page) Fig. 1.3 (continued) able to be completed without direct contact with a supervisor, while others will need direct response from supervisors prior to action. ## 1.6.2 Water Detection System Structure The WDS is comprised of several subsystems. The core system in the WDS is the main control element (MCE). The MCE acts as the brain in the feld by enacting orders from Earth, monitoring other subsystems, and linking the subsystems together. The other subsystems each perform specialized tasks (e.g., communicating with Earth, navigating the WDS, or collecting physical samples). However, all subsystems share a set of key features that the operators may interact with over the course of the mission. These features are shown in Table 1.2 and a diagram of the WDS–Earth link is shown in Fig. 1.4. ## 1.6.3 Example Issues System designers may be unable to anticipate every risk to system success; however, the Risk-Driven Incremental Commitment Model drives the designers to try to understand what risks are most likely to arise. Table 1.3 shows some example problems that could arise throughout the lifecycle of the WDS system, the risk of these problems occurring, the solution, and who handles them. Table 1.2 Key features built into each subsystem of the WDS Fig. 1.4 Diagram of the Water Detection System (WDS) and its connection to the operation center The WDS is designed to autonomously handle most issues that arise, but human interaction is required on a regular basis. Many of these tasks are simple maintenance and acknowledgement of warnings. For example, when batteries are low, the operator is required to acknowledge the low battery threshold. No action is required Table 1.3 Example problems faced by the WDS that require operator intervention other than clearing the notifcation. Occasionally, however, the WDS will face an urgent problem that requires human input. These scenarios are rare, so the operator has limited training in how to address the issues. ## 1.7 Principles for Design Based on the target system description, the example system, and the design process, we can provide an overview of the book as a set of design principles. These principles provide guidance on high-level concepts that the designers can use to improve the systems they create. We aggregate the most important design principles described in this book in Appendix 3. Though generally directed towards improving performance across the human–machine interface, these principles will often apply to the entire process of designing complex systems. #### Principle 1.1: Don't Assume the User to Be How You Think You Are One of the most important considerations for designers is to dispel the assumption that your users are just like you or how you think you are (we make the distinction because you might not think or work exactly like you think you do). Unless your user is a software developer, systems engineer, or astronaut, you will almost always need to adapt your design to meet the operator's system-related needs, capabilities, and wants (in that order). Designers often (perhaps due to the ready availability of themselves and the unavailability of example operators) make the risky assumption that the operator is just like them—this is almost never the case. It is therefore important to provide designers and engineers with the ability to consult users and other stakeholders throughout the design process. Methods for learning about users can include talking with them, watching them work, having them use your interfaces, reading their autobiographies, or watching movies about their work environments (whether documentaries or even fctional accounts). Each of these methods for understanding users will gather only a subset of the useful information; casting a wide net can reduce the risk of overgeneralization and improve the breadth of the knowledge gleaned from users. Understanding the operator enables engineers to mold the system design around the capabilities and constraints of its operators. Countless studies have shown that engineers often fail to understand their users. This knowledge is the foundation of user-centered design and leads to increased performance, fnancial savings, and safer systems (e.g., Bias and Mayhew 2005; Lewis and Rieman 1994; Pew and Mavor 2007; Ritter et al. 2014). #### Principle 1.2: All Design Choices Have Trade-offs—Don't Go in Blind Most design choices have trade-offs. This basic fact will provide engineers with diffcult decisions throughout the design process. For example, increased font size may increase readability by sacrifcing some valuable interface "real estate" and limiting the total amount of information displayed. Effectively resolving these diffcult design choices requires designers to use knowledge of the tasks and users to make informed decisions. Use of the risk-driven spiral model helps engineers make the best decision given the constraints by consulting with stakeholders and using what others have already learned. Designers will be presented with problems like this, both big and small, throughout the design process, and not every individual design choice is worthy of a full user study. For example, consider a system that requires operators to search for digital fles while performing other tasks. An informed designer may realize that recognition memory (i.e., "Is 'book\_manuscriptV47\_fnal.docx' the fle you are looking for?") is more robust than recall memory (i.e., "What is the exact name of the fle you are looking for?"). While searching for fles on a system, it is usually easy and familiar to point and click around a series of folders to fnd some item, as in the standard desktop operating system. Using a keystroke-based system (like a command line) might be faster, but typically will require more experienced users or more training. Stakeholders should consider which design would be best suited for their system needs, users, and tasks. As another example, consider a system that tasks operators with monitoring incoming pings and classifying them as friendly, hostile, or unknown. An informed designer will know that speed and accuracy are traded off when improving performance. Emphasizing speed will require sacrifcing accuracy (i.e., more errors), and the inverse is true as well. Stakeholders can use this knowledge to analyze how to reach an acceptable balance between accuracy and speed. Although ideal solutions are not always possible, designers can meet expectations by understanding the expectations for task time and error rate. Finally, almost any point-and-click system will use menu trees to support navigation. Many studies have explored how users' decision-making, reaction time, and error rate change in response to changing the menu design. The Hick–Hyman Law (Hick 1952; Hyman 1953) predicts that choosing between more options (e.g., fve menu choices vs. three menu choices) takes longer, but the menu is more likely to contain the correct choice. Signal detection theory shows a similar trade-off between hits, misses, false alarms, and correct rejections. When possible, engineers should make informed decisions about the trade-offs between outcomes caused by different design choices. #### Principle 1.3: Use and Test Multiple Designs When designing a new display or component, create and consider multiple versions. Get feedback on the possible designs from a source (or sources) that is as objective as possible. When you create a new display, particularly high stakes or main displays, you should consider multiple versions. Considering multiple versions of designs tends to lead to better designs at least in the tasks that have been studied (Dow 2011). The best objective source for feedback is often actual users' behavior. Research by Steven Dow examined the design process in the egg drop task. In this task, designers were given a set of standard materials and asked to design a protective cradle for an egg so it will survive a large vertical drop. Groups that designed more examples and that tested more often had reliably higher distances from which their eggs could be safely dropped. Dow argues that the benefcial outcomes seen from multiple designs will apply to other design tasks, and we agree. ## 1.8 Conclusion Throughout the design of an op center such as the WDS system and interface, the engineers' top priority will be the creation of a working product. However, engineers must account for the risks associated with all aspects of the project. Often, the risks associated with some module's reliability or function may trump the human element: human error requires a task on which to err. However, as the iterative design process advances, and the technology itself becomes more reliable, the human operator becomes more likely to be the point of failure within a system. Systems engineers will be neglecting a crucial component of their system if they do not account for the system's compatibility with the human operators. Although this process will have any number of constraints and variations in its implementation, the designers should be confdent that their system can be effectively used by the target population. The user interface should facilitate high performance without undue stress on the operators. Table 1.4 notes some questions that designers might have in mind when designing and implementing control rooms, op centers, and other similar systems. The next two chapters will review the psychology and human factors concepts and theories that give rise to the principles described above and should be considered to help answer the questions in Table 1.4. In the conclusion to this book, we will note how these questions have been answered. ## References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ## Chapter 2 How User-Centered Design Supports Situation Awareness for Complex Interfaces Abstract This chapter moves the discussion of how to design an operation center down a level towards implementation. We present user-centered design (UCD) as a distinct design philosophy to replace user experience (UX) when designing systems like the Water Detection System (WDS). Just like any other component (e.g., electrical system, communications networks), the operator has safe operating conditions, expected error rates, and predictable performance, albeit with a more variable range for the associated metrics. However, analyzing the operator's capabilities, like any other component in a large system, helps developers create reliable, effective systems that mitigate risks of system failure due to human error in integrated human–machine systems (e.g., air traffc control). With UCD as a design philosophy, we argue that situation awareness (SA) is an effective framework for developing successful UCD systems. SA is an established framework that describes operator performance via their ability to create and maintain a mental model of the information necessary to achieve their task. SA describes performance as a function of the operator's ability to perceive useful information, comprehend its signifcance, and predict future system states. Alongside detailed explanations of UCD and SA, this chapter presents further guidance and examples demonstrating how to implement these concepts in real systems. ## 2.1 Introduction The whole gamut of factors that contribute to the success of an interface is diffcult to describe within a single book, but the operator gives us a central focus. Just like any other component (e.g., electrical system, communications networks), the operator has safe operating conditions, expected error rates, and predictable performance, albeit with a more variable range for the associated metrics. However, analyzing the operator's capabilities, like any other component in a large system, helps developers create reliable, effective systems that mitigate risks of system failure due to human error in integrated human–machine systems (e.g., air traffc control). We identify some of the most signifcant factors that can affect operator performance and show how they can be used by engineers during their design of an interface. For a more comprehensive review, we recommend (a) Foundations for Designing User-Centered Systems: What System Designers Need to Know about People (Ritter et al. 2014) and (b) Designing for Situation Awareness: An Approach to User-Centered Design (Endsley et al. 2003b). This book offers design guidelines for optimizing the performance of the human component of the operation centers for asynchronous, autonomous systems. Figure 1.1 shows examples of the systems we are talking about like UAVS and satellites. User-centered design (UCD) provides the foundation for this task through basic tenets of its design philosophy. Designers can achieve UCD by designing for situation awareness (SA, explained below) in operators. Guidelines developed in these chapters will provide concise takeaways, while selected information on related cognitive mechanisms will provide context. Thus, this chapter will follow this logic. First, we describe the tenets of UCD. These provide high-level questions that engineers can apply to their system at any point in the design process. Next, the connection between operator performance and SA is explained. Performance levels of SA correspond with cognitive mechanisms used to perform a task. The fnal section describes the cognitive mechanisms and their infuences and offers design guidelines for ensuring compatibility between user capabilities and system interface. ## 2.2 User-Centered Design The operator is a component of the system just like the sensors or underlying code. High-performance systems will incorporate operator capabilities into their design. This requires creating a system that follows principles of user-centered design. Though UCD is often associated with user experience, Endsley et al. (2003b, p. 5) explain the difference between UCD and UX in underlying philosophy as follows: User-centered design challenges designers to mold the interface around the capabilities and needs of the operators. Rather than displaying information that is centered around the sensors and technologies that produce it, a user-centered design integrates this information in ways that ft the goals, tasks, and needs of the users. This philosophy is not borne primarily from a humanistic or altruistic desire, but rather from a desire to obtain optimal functioning of the overall human-machine system. The three primary tenets of UCD, shown in Table 2.1, describe the high-level goals of UCD. Each tenet is expanded over the next few pages alongside some explanation and examples. Table 2.1 The central tenets of user-centered design as summarized by Endsley et al. (2003b, pp. 8–9) To illustrate these tenets, consider driving as an example. Figure 2.1 shows a car's dashboard. With respect to Tenet 1, what are the primary and secondary goals of the user when using this interface? The design should refect the importance of each goal. While operating a vehicle, the primary goal is to arrive safely at the location; however, minimizing travel time is a salient secondary goal. Consider how the dashboard shown in Fig. 2.1 matches the goals, tasks, and abilities of a typical operator (or driver). The speedometer is large, detailed, and centrally located, which supports the operator's ability to quickly check vehicle speed, even during highway driving. This is the primary gauge that will be used while in motion, and thus is the most prominent feature in the display. The large tachometer provides instant feedback for operator input to the system, but with less detail than the speedometer. Broad markings and the red line provide simple indicators of system state. Engine temperature and fuel gauges are small and minimally detailed, with red lines indicating when direct action needs to be taken. The simple design suits their relatively infrequent use and their information complexity needs. What are the primary and secondary tasks that a user will perform on this interface? The design should refect the importance of each task. While driving, the primary task for this interface is checking the speed. The secondary task is monitoring the overall state of the vehicle. The speedometer has detailed markings to approximately match speed limits (10 km/h increments). The tachometer only provides broad details and a red line indicating an "unsafe state," matching the detail that a user requires for monitoring the state. With respect to the second tenet of UCD, the information in Fig. 2.1 makes the vehicle speed easy to perceive, interpret, and act upon. The other information for less important tasks is given less room. Where exact numbers are needed, such as miles traveled, this is provided as a number. Would a typical user be able to understand this system? Users and designers often have different skill levels and familiarity with the system. In the case of a car, the average driver is not a mechanic, so they often do not need detailed information Fig. 2.1 Image of a basic automobile dashboard. The full dashboard shows four gauges from left to right: tachometer, speedometer, fuel level, and temperature. From www.freeimages.com on most subsystems. An indicator light to check your engine may provide suffcient detail for a layperson who gets minimal value from additional details. Thus, Fig. 2.1 shows Tenet 2 in practice for the dashboard of a car. For the average driver, the check engine light provides only the necessary information to solve further problems and nothing more. With respect to Tenet 3, relevant information is provided to control the system. In this case, a user working through sequential information on a display expects the next area of focus to be on a path from left to right, top to bottom (as when reading). For the state of a car, the water temperature and gas tank level are suitably ordered. More complex interfaces may require a different order, and power plant control rooms often order the displays based on their location in the plant. In Fig. 2.1, if other information unrelated to driving the car was presented, such as distance from home, type of fuel in the tank, or brand of tire, the driver's ability to drive would be less well supported. If the prominence and organization did not match the driver's visual ability, for example, a less clear (or smaller) font, or dials presented in a different order, then the driver's performance could suffer. Finally, if the state of the car were less visible, or less appropriately matched to the frequency and importance of goals, performance would suffer. These tenets are not perfect, however, and do not always give clear guidance. Consider the display in Fig. 2.2. Here, the tenets do not provide direct guidance. The Fig. 2.2 Two ways to present display of an automated target identifer. Each design has trade-offs in operator performance that must be weighed based on the goals and priorities of the system. Image redrawn and modifed by authors. Based on a fgure from Banbury et al. (1998, p. 37) choice between these two designs must be based on the details of the goals and task priorities. If these are not known, they must be obtained from stakeholders (in the best case) or guessed or inferred (in the worst case). Together, the three tenets of UCD provide a foundation for how to frame the system design process around the goals, tasks, and abilities of the operators. The various other elements within a complex system have their own design philosophies or guidelines (e.g., modular design, minimal complexity, easy replacement of components). The human–system interface is no different. The tenets of UCD provide an underlying set of principles that should shape the design process for creating complex systems. Implementing UCD within complex systems requires a method for understanding and assessing operator performance during complex work. Endsley's (1995) theory of situation awareness flls this need by providing a framework for understanding performance and decision making. Describing the SA of an operator means describing the product of relevant cognitive mechanisms that are necessary to perform complex work like decision making and troubleshooting within an operation center. ## 2.3 Situation Awareness: The Key to UCD Human operators using complex systems must be able to correctly perceive useful information while ignoring or disregarding other stimuli. Situation awareness (SA) provides a framework for describing human performance on tasks ranging from driving an automobile to monitoring incoming cyberattacks. At a basic level, an operator demonstrating perfect SA knows which information around them is taskrelevant, what this information means for the present, and what this information will mean for the future. With these types of knowledge, the operator understands the current state and can effectively project their understanding into possible future states of the system. Describing an operator's SA performance uses three iterative stages. Though specifc performance benchmarks denoting each stage are derived from the tasks, the three stages of SA are typically known as (a) perception, (b) comprehension, and (c) projection. These are illustrated in Fig. 2.3. First, an operator must perceive the useful information from the task environment. Second, they integrate individual cues into a useful mental model of the current situation. Third, they use their model of the situation to predict likely outcomes based on their comprehension of the scenario. Figure 2.3 uses operation of an automobile to explain the types of information associated with each stage. Thus, operator performance can be improved through incorporating the tenets of UCD in system design. Improving the UCD of a system requires improving the SA of operators using the human–system interface. The system design will impact how well operators can develop and maintain SA during work. Interface design will affect how quickly and easily operators can advance to each subsequent stage of SA Fig. 2.3 The three stages of SA applied to task of operating a car. Figure redrawn and modifed by authors. Based on a fgure from Bolstad et al. (2010, p. 4) performance and how accurate and complete the operator's understanding is at each stage. Similar to shifting gears in a manual car to increase speed, the stages of SA progress on a continuous scale where competency with lower levels of SA is required to advance to the next stage. The stages of SA provide a framework for assessing performance and identifying task and interface factors that can moderate SA performance. Progression through stages of SA will be impacted by operator characteristics (e.g., fatigue, personal capabilities), environmental effects (e.g., distractions), and task-related factors (e.g., cognitive resources required, task types, complexity; Boff and Lincoln 1988). Each stage requires signifcantly more resources (e.g., knowledge, information, time) than the previous. Stage 3 SA should not be expected as the norm for every operator or every task; however, it is the most useful. Next, we describe the stages of SA in more detail and provide principles for design based on using SA as a metaphor for work in op centers. These principles are derived from Endsley et al. (2003b) and are applied by us to apply SA to the design of op centers. We include motivating examples for each stage. Tasks surrounding aviation were the original focus of SA research before it expanded to include a variety of complex tasks. During this discussion, we will describe the frequency of aviation disasters caused by critical errors in each stage of SA. These error rates refer to errors in common aviation tasks for pilots, air traffc controllers, and other aviationrelated jobs, but it would be reasonable to assume that similar results would be found across a variety of op centers. ## 2.3.1 Stage 1: Perception Perception is the most fundamental aspect of SA. During the common tasks within an op center, operators are likely bombarded with information. In most cases, space and cost in op centers will be at a premium, leading to operators with varied tasks across multiple displays. Each of these displays could be presenting tens or hundreds of data points, graphs, or other useful features, meaning that a major component in skilled performance could be simply knowing where to look and when. The situation and signal content can determine the best course of action regarding how and when to respond to a signal (if at all). Operators with Stage 1 SA will demonstrate the ability to detect important signals while discarding irrelevant ones. Given perception's fundamental role in an operator's work, it is unsurprising that perceptual issues account for about 75% of errors in common SA work (Jones and Endsley 1996). Causes of Stage 1 errors may be attributed primarily to human failures (e.g., attentional failure, misinterpretation of a signal), system failures (unclear or missing information), or some combination of the human and system failure. Some design principles related to Stage 1 SA are shown in Table 2.2. The principles can be summed up as follows: task-relevant information should be readily available, easily interpretable, appropriately prominent, and simple enough for the typical user. For example, in the WDS (introduced in Chap. 1 and explained in detail in Appendix 1), a display can indicate that the battery will be unable to charge at the rover's current position and the rover will need to relocate. The interface must clearly convey this information for the operator so they can instigate a "move" command before the battery is too low. The interface should provide clear signals of the system state like a commonly used alarm icon (available) with a text description (interpretable) that fashes (appropriate salience) until the operator schedules the appropriate command (simple). While it is somewhat common practice to rely on unlabeled "self-explanatory" icons (i.e., for alarms), designers concerned about reducing risks of confusion, and errors will support the visual design with liberal use of textual labels. Words in interfaces are often underused but are more easily interpreted than symbols when used alone (Chilton 1996). The principles in Table 2.2 provide a framework for ensuring the interface can effectively convey useful information in a manner that is useful to the operator. This means ensuring that the value and salience of each piece of information is appropriate, actively drawing attention to important signals, and minimizing the quantity and salience of extraneous stimuli. The second principle in this area is to make the information interpretable by using intuitive, sensible designs. The third principle extends the frst two by promoting a hierarchy of signal importance to ensure that the signals perceived by the operator are the most useful at any given time (or at least that non-useful signals are relatively muted). The fourth principle deals with Table 2.2 Design principles related to Stage 1 SA the inherent limits to human cognition. While these limits tend to be loosely calculated, designers can follow this guideline by working broadly to reduce complexity across the system whenever possible. As an example, reconsider the car dashboard shown in Fig. 2.1. Several design features facilitate Stage 1 SA during typical operation of the vehicle. Compare the prominence of the speedometer and tachometer to the temperature and gas gauges (Principles 2.1, 2.2). Operators likely update their mental model of speed and engine performance every few seconds, but only check the temperature and fuel levels if something is going wrong (Principle 2.3). Taken together, this design takes steps to limit or reduce the availability of unnecessary or distracting information (Principle 2.4). While the design of the dashboard could likely be improved, this example shows how simple design changes like changing size proportions can support Stage 1 SA. The dashboard design also supports monitoring for infrequent, but critical, alerts like low fuel levels. The fuel level indicator provides two different signals when fuel reaches dangerously low levels. First, the fuel level gauge displays the current fuel level compared to a warning level. This allows the operator to quickly assess the current fuel level and determine whether action is needed (i.e., adding fuel). Even outside of warning situations, the operator can maintain suitable awareness of the fuel level and plan accordingly. If the operator fails to add fuel before reaching the warning level, the second alarm signal will trigger: the fuel level icon of a gas pump will glow yellow. This provides a second chance for the operator to respond to the situation if the frst chance (fuel level indicator) fails, and only appears when fuel is dangerously low. Newer cars will even sound an alarm or, better yet, vocalize the alarm information. Altogether, the fuel level gauge supports Stage 1 SA by making the information available, salient, and appropriately designed to mitigate risks to system failure (i.e., running out of gas in the middle of nowhere). For another example, consider the WDS introduced in Chap. 1. When below a certain power threshold, the dashboard interface displaying the battery information will continually fash a red symbol, indicating the risk of total power failure for the system. If this alert continues until the battery is charged, the signal will waste the operator's attention and cause unnecessary distraction. Why does the signal remain prominent, even after the solution has been implemented? Once the solution process begins, there is no need to draw attention to the signal until additional information is received. The signal's visual appearance should be able to be muted until another update is needed. This principle has further implications for the details of displays. It suggests eliminating or suppressing unnecessary signals and merging compatible signals. Simplify complex signals. For example, an interface showing the overall WDS status may include orientation, geographic information, battery level, and other information. These parameters are monitored by operators for unexpected changes; however, excessive details increase workload by increasing the amount of visual clutter. Designers should strive to optimize the complexity and detail when possible, which in many cases means reducing those factors. If you know operators only check the approximate orientation (i.e., NW, S), then that's how orientation should primarily be displayed. And if the detailed heading information is still required to be shown for occasional use, then the salience of that information could be reduced (e.g., reduce text size, use muted colors for font). The fourth principle in this area is to work with the limits of human cognition and perception. Human cognition has natural limits in how much it can process at once. Work around the limitations by reducing complexity and workload of the task. For example, a status update for the WDS may include hundreds or thousands of events in a data log that accompanies the basic system status report. Reserving a space on the interface to indicate critical or alarming events (e.g., imminent power failure) while hiding data related to non-important (or typically non-important) updates will reduce the amount of information necessary for the operator to perform the most useful tasks. As another example, consider a system that is rarely interacted with during normal operations. The interface simply provides a status that is checked hourly by an operator. This interface was initially expected to be part of a multiple-monitor display for a seated operator, but now it is checked while standing several feet back. Now the operator must lean in or squint to read and understand the information. Consider physical aspects of how the operator uses the system. An operator sitting at a desk in front of the screen can effectively monitor more dense signals than someone 5 feet away. Ideally, the perceived details of an interface will smoothly transition as an operator views it from different distances. While the people building these types of systems should typically avoid overly bold designs, there are still useful lessons to be learned regarding how aesthetics can affect operator performance. Books on visual design of interfaces can provide more information in this area (e.g., Kosslyn 2007; Tufte 2001, 2006). ## 2.3.2 Stage 2: Comprehension The second stage of SA involves synthesizing Stage 1 cues into a useful mental model of the situation. A practiced operator will purposefully seek out patterns from various stimuli and form a holistic view of the situation based on their experience with the task and the information presented. Errors arising from comprehension failure account for about 20% of errors (Jones and Endsley 1996). Stage 2 errors are often attributed to misinterpretation of an information set, failure to maintain all the necessary information in working memory, misuse of a mental model, or overreliance on default settings (e.g., failing to check a status hidden behind a submenu). Some design principles related to Stage 2 SA are shown in Table 2.3. As an example of the frst principle, the interface that provides the WDS status information may have a variety of information presented on it using textual and visual signals. Icons can help reduce text or provide a more grid-like design, but should only be used when the operator understands the meaning (so make sure that the operator understands the meaning through culture, training, pop-up names, or other means). Principle 2.5 Actively design the system to prevent misinterpretation of signals. Signals should be unambiguous, consistent, and instantly recognizable Principle 2.6 Consider how the actual tasks will be done by the operators. If operators will be expected to multitask, then build in features to accommodate this fact Similarly, familiar symbols should have familiar meanings. Using an "X"—particularly a red "X"—should typically indicate that something will "close," "exit," or "cancel." Red and green follow cultural norms of stop/exit/bad and go/continue/ good, respectively. The Apple Design Guidelines1 give an example set of such guidelines. The second principle is to consider how the actual tasks will be done by the operators. Interruptions and task-switching are major sources of error. If task interruptions are common, designers should account for their effects in their task analyses for the system and seek to mitigate their negative effects on task performance. These design features can include the ability to postpone the next task so that the current task can be completed, or to remember the state of the suspended task until it can be returned to. Sometimes even non-digital solutions can work; in a control room, one solution could be to simply include a pad of paper for note-taking (Trafton et al. 2003). As an example, operators may have to multitask while monitoring the WDS. The WDS status interface provides many different pieces of information, but the operator will typically not have any issues responding to routine events. However, once they need to respond to some new situation, they must split their attention between the normal monitoring and the new task. This could lead to the operator missing an important warning. The system could support this task requirement and reduce risk by providing a simplistic view of critical information during times when the operator may be splitting attention across multiple tasks. When an operator pulls up a subsystem view alongside an overall status view, the overall status could become less detailed while increasing the salience of signals indicating new changes. Or alternatively, operators could be prompted to use simpler methods for tracking system state, such as a pad of paper or a sticky note on the screen, which could allow the operator to "save" the partial state information prior to dealing with an interruption. Further information on how cognition is used to comprehend a situation is available in Endsley's work (Endsley et al. 2003a, b) and other books on human–computer interaction (Krug 2005; Ritter et al. 2014). <sup>1</sup> https://developer.apple.com/design/human-interface-guidelines/ ## 2.3.3 Stage 3: Projection The third stage of SA is achieved through projecting the model of the situation into possible future outcomes. For example, an air traffc controller could anticipate a dangerous situation based on how two aircraft are likely to maneuver while changing course and act to avert the future incidents. Though diffcult, this type of expertise is essential for high performance in some complex tasks (Endsley 2000). Stage 3 failures account for about 3% of errors in aviation, but the complexity of Stage 3 SA makes generalizable causes of error diffcult to isolate. General causes may include overtaxation of mental resources, insuffcient knowledge of the domain, or overprojecting current trends (Jones and Endsley 1996). This type of expertise is diffcult to plan around for the engineers during the early design stages, and thus will be given less focus in this book. Obviously, systems that help predict the future of object or systems would help operators. For example, supporting Stage 3 SA could be as simple as including trend lines showing system state over time, or as complex as automated calibration of signal strength to predict upcoming alert states (Tufte 2006). One of the most effective ways to design for Stage 3 SA is by eliminating barriers preventing Stage 1 and 2 SA from being effectively supported. Thus, designers are advised to focus on solving issues with perception and comprehension before specifcally addressing methods for improving an operator's ability to project into future states. However, further information about supporting projection can be found in Endsley's work (Endsley et al. 2003a, b) and work on mental models (Besnard et al. 2004; Kieras and Bovair 1984; Moray 1996; Ritter et al. 2014). ## 2.4 Summary: Cognitive Mechanisms for Situation Awareness The three stages of SA provide a broad classifcation for the performance of operators during complex tasks. This chapter only briefy describes SA. This overview gives engineers the tools needed to consider how SA applies to the systems they design. In the next chapter, the cognitive mechanisms that drive operator performance are described and connected to SA. This chapter briefy covers signifcant cognitive mechanisms used in SA as a way to describe and summarize them. These mechanisms and their role in SA get more comprehensive coverage in Chap. 3. We explain them here because these cognitive mechanisms can be simulated in a computer (Anderson 2007), but can also be simulated in the designer's head to make predictions about how operators use the system. Figure 2.4 shows these mechanisms as they are implemented in the ACT-R cognitive architecture (Ritter et al. 2014, Chap. 1). These components can be seen as distinct subsystems with semi-independent operations. To learn more about ACT-R, Fig. 2.4 A schematic of the components of a computational model (ACT-R) of the human operator. (Figure used with permission from Ritter et al. 2018; Fig. 3) Ritter et al. (2018) review the state of research using ACT-R and other cognitive models. As shown in Fig. 2.3, the process of achieving situation awareness often starts with perception, the intake and processing of competing sensory cues (or signals) into usable information. In this approach, perception does not necessarily lead to detection of a signal or to understanding because the perceptual process requires attention from cognition. Attention, in this case, means that select information is targeted by the system. Cognition, the central process, directs focus on the taskrelevant information while ignoring or not processing the rest. Attention is a limited resource that must be distributed across appropriate features. Attention is probably best seen as an active process of directing cognitive resources rather than a single buffer responsible for passing information. Top-down attention is goal-directed towards some feature(s) based on the goal while avoiding focus on distracters (e.g., monitoring speed and position but ignoring billboards while driving). Bottom-up attention is driven by the common features that indicate activity (bright colors/lights, motion, and others). Memory is used to perform the task, recruited from the declarative memory buffer or activated from long-term memory (in ACT-R, in the declarative buffer and the goal buffer), which might be called working memory (WM), which operates as the "RAM" for cognition by storing and manipulating information chunks for short periods. This stored information has to be maintained through use, manipulated, and stored in long-term memory, or it decays and is lost. Human memory is more similar to old drum or plated wire memory, which needed to be continually refreshed, than it is to current solid-state RAM, which can sit without use and without decay. WM is more than just a singular "catchall" for temporary information storage. The current theory of working memory has established at least two major subsystems, the visuospatial sketch pad and the phonological loop, which exclusively hold visual and verbal information, respectively (Baddeley 2012). Each subsystem operates semi-independently to store and maintain information for near-term use. One beneft of these distinct storage types is an improved ability to multitask when we distribute the cognitive operation across multiple WM stores. Dual-task activities can be performed well if each task uses only, or mostly only, a singular WM store. For example, it will be easier to remember a set of numbers while observing a scene in a play than while solving math problems. The operator's mental model is the operator's internal representation of an external situation. Their mental model provides the framework that they use to process information related to the task. This model is stored in memory, which means it can be learned, or partially forgotten, and might not match the designer's representation used to understand the system and to create the interface. The operator's mental model of a situation provides the tools needed to handle large amounts of information. They use their experience from long-term memory to scaffold the intake of new information, noting what to pay attention to, what to discard, and what to remember for a given situation. Mental models also include what to do in a situation. Thus, situation awareness, the awareness of the state of the world, what is happening, and what will happen, is based on an operator's mental model and its used by a set of mechanisms similar to what is in Fig. 2.4. This approach, when applied to op center design, suggests that each stage of the operator's processing and response is important for a successful system operation. The operator needs to be able to see and process the stimuli. They need to be able to have attention and time to understand them, and the ability to acknowledge that the stimuli are important. They need to have an appropriate mental model in which to relate new information to previous information and current goals. They need to know what to do, and how to respond. And they need the world's state and a good mental model to predict what will happen in the world. Situation awareness thus provides a way to organize a designer's model of the operator. It makes strong suggestions about design when combined with knowing the operator's capabilities, their tasks and task priorities, and their mental model of the world. This model accounts for both the long-term learning and mastery of the system and the ongoing and evolving model of what is happening at any point in time. The next chapter explains these components in more detail to help a designer understand how an operator might run and apply their mental model. 34 2 How User-Centered Design Supports Situation Awareness for Complex Interfaces ## References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ## Chapter 3 Cognition and Operator Performance Abstract Developing systems that foster situation awareness in operators requires that stakeholders can make informed decisions about the design. These decisions must account for the operator's underlying cognitive processes based on perception, comprehension, and projection of the system state. This chapter reviews the core cognitive processes responsible for monitoring and responding to changes in system state. Operators must perceive information before they can act in response, and the interface design affects operator accuracy and speed via known mechanisms (i.e., effects of color on visual search time). Perception of key information also relies on how the operator thinks during tasks, and certain design choices can support better attention control and detection of signals. After perceiving the information, operators also must comprehend and interpret the information. Design guidance and factors related to supporting comprehension are presented alongside explanations of how cognitive load and working memory affect the operator's ability to develop and maintain a useful mental model of the system. This review of cognitive mechanisms gives designers a strong foundation to make informed decisions ranging from choosing an alarm color to assessing how much information should be on screen at once. ## 3.1 Introduction This chapter explains in more detail the primary cognitive mechanisms used by operators to perform their tasks. This chapter should help designers have a better mental model of operators. These details should help a designer understand how an operator does their tasks and thus support the operator better. In this approach, based on the cognitive architecture shown in Fig. 2.4, cognition can be described as an emergent phenomenon arising from a collection of mechanisms. The mechanisms are components of an information processing system in the same way that a computer has components. The component mechanisms can be described in isolation (e.g., visual processing of an object) with a great degree of useful truth. However, it is important to understand that this is a practical consideration. In truth, cognition relies on an extremely complex, highly interconnected neural system. © The Author(s) 2021 37 J. D. Oury, F. E. Ritter, Building Better Interfaces for Remote Autonomous Systems, Human–Computer Interaction Series, https://doi.org/10.1007/978-3-030-47775-2\_3 This chapter explains these mechanisms in detail to help a designer. The mechanisms discussed here include visual perception, attention (which is perhaps emergent from other system interactions), memory, and learning. In each section, we note design principles to summarize the results and aid design. ## 3.2 Visual Perception The most basic level of cognition for operators is the perception of stimuli. Whereas we may be able to receive signals from a variety of sources, visual stimuli provide the proportional supermajority of signals. Auditory comes in second, followed in a distant third by tactile (which does not appear to be used nor needed currently in most control rooms). We will follow this natural system order in our analysis. Thus, we will primarily focus our discussion on visual perception. ## 3.2.1 Visual Processing Understanding the nuances of visual processing enables system designers to build their interface around the natural capabilities and limitations of the operators. At a basic level, visual processing is the process of capturing light on some visual sensor and transmitting this information to the processing system. For many robotic systems, this is a relatively straightforward process where information only fows in one direction. In contrast, human processing is a bidirectional process including feature detection, goal-directed attention, pre-attentive assessment of stimuli, and active interpretation of the signals. This complex system allows us to make a sensible, coherent world out of small snapshots of information without the need for detailed processing. While humans may excel at particular tasks like pattern detection, we also can be easily tricked by unconscious misapplication of visual processing heuristics (e.g., visual illusions, misrecognition, not seeing target objects). While some sources of errorful behavior can be inhibited or corrected through conscious effort, others are essentially refexive actions without any reasonable method for self-regulation. A classic example of our failure to inhibit automatic processing is the Stroop task (Stroop 1935). The task is simple. A subject is presented with a color word (e.g., red, blue, yellow) written in one of those same colors. The task is to name the color of the ink. The experiment has two conditions, congruous and incongruous. When congruous, the ink color and word will match (e.g., "red" written in red ink). When incongruous, the ink color and word will not match (e.g., "red" written in yellow ink). This task seems simple in the congruous condition, but when the incongruous condition is tested, and the word and its color differ, the subject will typically stumble through responses, be signifcantly slower, and make many more mistakes. Once we learn how to read, we simply cannot inhibit the natural response to read text. The mechanistic explanation is that the reading skill is practiced so much more than the naming skill; thus, the reading skill must be suppressed to name the color. Unless some cognitive effort is used to direct attention, the "over-practiced" reading skill will force out the less-practiced skill when both use the same mechanisms. A more comprehensive overview of low-level visual processing as well as additional resources can be found in the chapter "Behavior: Basic Psychology of the User" (Ritter et al. 2014, Chapter 4). ## 3.2.2 Color Blindness Color blindness is a particularly salient concern for designers due to its prevalence among the population. For the Western population, about 8% of men and 0.5% of women have some form of red–green color blindness. This causes affected individuals to have diffculty differentiating red from green. Individuals may also have blue–yellow color blindness, or even total color blindness, but these are signifcantly more rare than red–green color blindness (Ritter et al. 2014). There are many several forms of color blindness, based on the specifc defciency in the visual system, but the general design recommendations that alleviate their effects are the same. Good design will avoid using only color as a signal for an operator. Instead, the design should incorporate multiple signals into a cohesive message for the operator. For example, an important alarm could fash bolded text information, have red coloring, and use textual indicators like exclamation marks to ensure that the message is clear. Thus, better designs will dual-code results. That is, meaning will not just be encoded by color but color and font, or line thickness and name, or line type and texture. Dual-coding stimuli makes them faster to be recognized and discriminated (Garner 1974). It may be useful to check designs for adherence to color blindness design standards. There are tools online to show how color-blind individuals perceive images and interfaces.1 They typically take a URL or image fle and show how color-blind individuals would see it. Given the prominence of color blindness among the general population, dual-coding signals and ensuring color-blind compliance would be well-advised for any system that requires human operators. ## 3.2.3 Visual Search The visual system can be broadly broken up into two subsystems based on their role. The eye handles stimulus detection, and the brain (in specialized regions) handles stimulus interpretation. Stimulus detection occurs within the eye, but the <sup>1</sup> (e.g., https://www.toptal.com/designers/colorflter/) process itself is driven by a combination of goal-directed attention from the mind (top-down) and automatic processing of salient features (bottom-up). Top-down and bottom-up directives guide the visual processing and integration of the environment that occur during visual search. This confict between top-down and bottom-up visual processing means that designers should consider how their design interacts our natural visual mechanisms. Visual search of the information displayed on an interface is a core activity for operators, regardless of the task. As their attention is oriented to the task at hand, the operator will need to comprehend the information presented on any given interface. Visual processing is an intermittent process in which our eyes are constantly alternating between saccades (rapid eye movements to some feature) and fxations (resting moments of information intake). What we perceive as a continuous visual experience is actually an intermittent series of fxations that are unconsciously aggregated into a coherent, though not necessarily accurate, mental model of our surroundings (Irwin et al. 1988). During fxations, feature detection relies on distinguishing target features from distracter features through pre-attentive visual processing (Healey and Enns 2012). This summary of vision as being active can be contrasted with folk psychology and early understanding of vision where humans were understood to see and understand the whole display at once. We now know that the eye must search for information actively on the display and often refresh what it sees (Findlay and Gilchrist 2003). During complex tasks that require visual search, both bottom-up feature recognition and top-down goal-oriented activity infuence the performance of the operator at fnding that information. While top-down directives lead visual search towards a certain set of features, our eyes are unable to fully inhibit the bottom-up feature detection. Given the effects that distracting features can present for operators, designers should understand what types of visual features draw people's attention and the role of higher-level graphical organization. The best systems engineers and designers will have a theory of how users will scan displays, fnd the salient information, and understand it. ## 3.2.4 Pre-attentive Visual Processing Once an operator perceives the signals presented by an interface, the visual processing system immediately begins working to form a coherent mental model of the scene. Cognitive limitations on information processing prevent humans from scanning, processing, and understanding every individual signal within the visual feld. Instead, we have developed a complex pattern-matching system that reduces workload without (usually) negatively impacting comprehension. There are two main processes that occur during the early stages of visual search. The frst is pre-attentive visual processing based on relatively simple features of the objects. Figure 3.1 shows examples of the types of features that are easily and immediately detected during visual search. The common element across these Fig. 3.1 Examples of pre-attentive visual features. (Adapted from Healey and Enns 2012, p. 1172) examples is the contrast between features. When objects vary in orientation, length, or size (compared to other objects in their environment), they are identifed and distinguished much more quickly than other objects. Easily distinguished visual features are more salient to the operator, particularly when the operator is distracted or overworked. The contrasting features shown in Fig. 3.1 vary in their salience. Just by glancing across the examples, we can notice a difference in how rapidly we acquire the target stimulus among the distracters. The target feature for orientation is easily discerned, while the target features for lighting direction and length take slightly longer to be recognized. Designers must consider the salience of the signals they will present to the operator and allocate the most salient cues to the most important differences. The second major process of early visual processing is the grouping of individual features into shared, higher-order visual structures. This is known as Gestalt grouping or Gestalt theory (Chang et al. 2002; Moore and Egeth 1997). Just as particular features are distinguished individually, sets of features are organized into visual structures to be further processed by the viewer. This organization in the scene enables the viewer to maintain a mental representation of a coherent set of distinct objects drawn from the information-dense world. Just like the processing of preattentive visual features, Gestalt grouping is an involuntary processing step that shapes how a person perceives the world around them (Moore and Egeth 1997). Gestalt theory encompasses a family of related psychological principles of perceptual organization used to describe common instances of visual integration. The literature on this subject is varied, and as such, the specifc principles can often be described in multiple ways depending on the situation or researcher. Though not exhaustive, Fig. 3.2 shows seven of the most common examples of Gestalt principles affecting how we aggregate component pieces of a visual image. These principles can be used by a designer to group information together or separate different subgroups appropriately. Fig. 3.2 Common examples of Gestalt principles affecting image perception. (Revised from Ritter et al. 2014; Figure 4-15) Even without other factors affecting visual processing, Gestalt theory can serve as a useful framework for analyzing and improving the design of an interface. Chang et al. (2002) demonstrate how Gestalt theory can be used to guide the redesign of an electronic learning tool. During their background research, the authors identifed a subset of the many Gestalt "laws" from prior research and used these as the basis for their redesign process. The redesign process described by Chang and colleagues provides a useful exemplar of the methodology; however, they did not collect the empirical data necessary to provide a detailed analysis of how their redesign affected interface performance. ## 3.2.5 Summary of Visual Perception and Principles Nearly everything on the interface is a signal or feature. Designers should assess the importance of each signal as well as the salience associated with it. The theories in this section provide ways to make the combined operator-interface system work more reliably and, thus, reduce the risk of total system failure. To make signals recognizable, designers can change the hue, make it fash, increase the size, or use the pre-attentive visual features shown in Fig. 3.1 to modify the salience of the information. The inverse is also true. For irrelevant features (at least for the current task), ensure their salience is appropriate by modifying their visual representation to make them less apparent. If an operator does not perceive an alarm or signal directed their way, they have no way of knowing there is an issue, or even that they missed an alarm at all! Creating a mental model requires unconscious assumptions about the world. Do not assume that the operator will eventually realize that they must attend to a minor signal or remember to look at something; help them. It may be appropriate to test the interface for color blindness compatibility. Where colors cannot be changed, one could test the users to support reconsidering changing colors, or to fnd other ways to support color-blind users. Gestalt principles give engineers the ability to predict, and thus improve, how operators will perceive the interface and its functionality. Designing the system layout around these principles can ensure that the engineer's intentions are clearly conveyed to the operator. To summarize how to use results from visual perception in design, we present a few design principles related to vision. #### Principle 3.1 Designing to accommodate color blindness will solve multiple problems at once The prevalence of color blindness among the general population means that accommodating color blindness should be the default plan for high-stakes systems. Presenting information with multiple signals and modes can help ensure the message is clearly received regardless of the operator's color perception, and it will lead to faster detection of key signals. Principle 3.2 Colors must be used sparingly, used consistently, and should be reserved for critical information Color can be recognized and interpreted much more quickly than a complex signal, but overuse reduces the effectiveness. If possible, follow these rules: use no more than four different colors, adopt a dull screen as background, and reserve specifc colors for specifc signals. Thus, ensure that color provides a valuable signal to the operator through purposeful use of specifc colors to emphasize critical information on an otherwise dull interface. Often, color can be a distracter just as easily as a signal if the colors are overused or misused. Three specifc examples are shown in Figs. 3.3, 3.4, and 3.5. Designers must consider how each color used in the system will be interpreted by operators. Figure 3.3 shows a relatively dull interface that can be quickly scanned to identify which system processes are active without any distracting signals. Connecting lines between components (light yellow) are easily distinguished, but the reduced saturation demotes their importance during typical use. Color is often a major factor used within an interface to encode signals with meaning. Color use will usually use pairs or sets of colors to provide a categorical piece of information for the operator. Green, yellow, and red can indicate the system status on a range from healthy to critical failure. Blue can represent active pumps for a liquid, while gray shows inactive. Color is a valuable signaling method for typical operators, but designers should ensure that their design has multiple signals indicating critical information. Figure 3.4 shows an example of how color can be used to highlight critical information (Ulrich and Boring 2013). The use of color within an interface should be considered as a scarce resource. On a completely plain background, one color can be extremely visible, but each new color and new use of a color reduces the salience of that signal. The information in Fig. 3.4a uses a blue line to indicate the current level, which is then compared to "safe" levels on the right side (red and green lines). Fig. 3.3 Labeled example of interface with dull color overall, allowing the green "active pump" and red "critical event" signals to stand out. Figure redrawn by authors and modifed from Ulrich and Boring (2013) Fig. 3.5 Incremental improvement of power level indicator. Final product can be quickly referenced for general status and examined more closely for detailed information like voltage and time remaining For example, the gauges shown in Fig. 3.4a may be unable to provide color-blind operators with enough information to ensure system success. Figure 3.4b shows a revised interface that would be better suited for all users. Though the second gauge sacrifces some contrast between the safe and dangerous system states, the thick black line and arrow indicating the current level reduce the risk of color blindness leading to operator, and thus system, failure. #### Principle 3.3 Make text with readable fonts, use no more Than three font types, use fonts of proper sizes, and use simple, short text strings Reading from screens tends to be slower and more diffcult than print-based reading. This may be due to the difference between projective and refective light or due to pixel density. Researchers have studied the effects of screen-based reading quite extensively. They consistently fnd that reading from screens takes about 10–30% longer, leads to increased errors, and fatigues the user more quickly than print reading (Ritter et al. 2014, pp. 208–210). Many operators will not be trained to differentiate font types, so use different fonts sparingly and be cautious about using font type as an important signal. Improve readability and comprehension by using readable, simple fonts. Ensure font size is appropriate for the expected viewing distance. Concise text, accompanied by a symbol or icon, will be faster than a description and more easily interpreted than an icon alone. Designers should thus avoid using unnecessarily "fancy" fonts and settle on simple, effective presentation of the key information. In general, long strings of text should be avoided. They can be replaced with symbols and bullet points or, at the very least, augmented with emphasized words to make scanning easier. Figure 3.5 shows an example of improvement. #### Principle 3.4 Ensure signals indicating missing information are clear and obvious Operators rely on gathering and interpreting information to make key decisions. Uncertain or missing information can affect performance through incorrect assumptions by operators. Missing information from a sensor or system can be a signal to the operator about the situation, but this is only possible if the operator is aware that the information is missing. When operators do not realize that some information is missing, they may rely on their base assumption of normal operating conditions. This is called the normalcy bias and can lead to potential disaster. For example, a pilot operating a plane in cloud cover with malfunctioning terrain sensors can respond differently if aware of the missing information. If aware of the issue, they could climb to a safe altitude regardless of any "true" obstacle. If unaware, they may crash after assuming they were on a safe trajectory. This type of catastrophic failure is so common that it has its own name, CFIT, or controlled fight into terrain. As an example for the WDS, signals indicating success for a repeating procedure could be represented as a simple binary response: success or failure (1a and 3 from Fig. 3.6). The interface design in Fig. 3.6 may allow operators to quickly see when the last test occurred and provides an intermediate signal for a missing self-test. If the update schedule is known to vary by 30 min, this could lead to many false alarms if a missing self-test at the exact due time qualifes as a critical failure. These additional states added to the design give operators a signal to be in a "ready" state to respond to a critical failure. #### Principle 3.5 Arrangement of screen components should be useful, consistent, and close Whether designing the full system interface with multiple objects or creating the objects themselves, limit the distance between signals that are commonly used together. This means having a theory of how the interface will be used and using the task analysis, operator knowledge, and characteristics to design the interface such that the information and signals used for the same tasks are near each other. This principle is implied by the Gestalt principles. Fig. 3.6 The role of color to represent missing and aging information As an operator scans the system interface during typical monitoring tasks, they will be generally searching for alarms, alerts, or any sign indicating a potentially risky situation. The task analysis should provide a summary of the tasks, their importance, and their frequency. Checking systems with distant components (measured as travel time through the interface) requires more time and effort to perform well. Additionally, upon identifying an alarm, operators often will search for signals that confrm the veracity of the alarm. Grouping related components together makes this easier, reduces strain, and increases their ability to search for information. Grouping and arrangement should also attempt to follow consistent patterns both visually and semantically across multiple displays. The design guidelines in Appendix 3 (specifcally in A3.3: Visual Feature Index) provide guidance about the terminology, signifcance, and heuristics that designers should use when building these systems. ## 3.3 Attention Visual perception is broadly described as the integration of information through the feld of vision. However, this does not account for how useful signals are isolated from the noisy environment around them. Attention is the "spotlight" that makes a set of stimuli more active or relevant than the rest of the display. As operators are presented with a constant array of information, an executive control system in the mind is directing attention towards features or items in that set of information. A crucial feature of attention is enhanced acuity for the target of interest at the expense of awareness of peripheral stimuli (Ritter et al. 2014, p. 139). The shift in focus from one target to another can occur due to the salience of certain features, perceived relevance to a particular goal, or an active process of cognitive control. In this section, we will frst discuss the basics of the underlying mechanisms of attention and how task-switching affects operator performance. Next, we will describe the causes and implications of limited attentional resources and the attrition of attention. Attention plays a crucial role in visual perception by providing a mechanism for isolating specifc features of interest. Visual perception involves making sense of a world with too much information present; attention is the tool for "working around" this natural limitation. Attention provides guidance for, though not total control of, the sequence of eye saccades and fxations during goal-directed search for visual features. The interaction between visual perception and attention is moderated by cognitive control (e.g., goal-directed behavior) and aspects of features in the visual feld (e.g., salience). The interaction between these two systems can affect performance by altering the usage of "cognitive resources" during a particular task. For example, inhibiting a response to look at a fashing light requires active control of visual search, and thus attention. The skill with which a user can inhibit these responses is governed, at least in part, by their working memory capacity (Unsworth et al. 2004). The inverse is true as well: an extremely salient signal will require fewer cognitive resources to detect. ## 3.3.1 Attentional Vigilance The role that attention plays in cognitive tasks cannot be overstated. Although we have primarily been describing the role of attention on visual processes, attention plays a central role in both internal (e.g., problem-solving, goal sustenance) and external cognitive mechanisms (e.g., visual search). The act of maintaining attention on a task is called attentional vigilance, or just vigilance. Tasks that require vigilance are characterized by the need to maintain attention over an extended period while attempting to detect target stimuli without responding to neutral or distracting stimuli. Performance loss is often ascribed to a vigilance decrement, or the performance decline that occurs over a period of active monitoring. Tasks that require vigilance are extremely common for operators during their work in op centers. Sustained attention on a task can be impaired by several factors. First, the salience of the goal signals directly affects the decay rate of operator performance due to the vigilance decrement (Helton and Warm 2008). Increased working memory load leads to worse performance on vigilance tasks. If an operator needs to remember other tasks or keep other information in working memory, they will have a higher cognitive load (Helton and Russell 2011). Depending on the type of information being remembered, the impact on performance may be reduced. For example, listening to a supervisor speak (verbal) while monitoring trends on a graphical display (visual) is easier than listening while reading text (both verbal) (Epling et al. 2016). The ability to maintain attention over minutes or hours is also affected by the time of day and the natural circadian rhythm that is driving the operator's sleep schedule. The impact of sleep and restfulness on performance varies by the task characteristics. Discrete, active motor control tasks (e.g., tilting a platform to roll a ball towards a hole) seem to be less affected by sustained time awake (Bolkhovsky et al. 2018). However, the biggest concerns should be for monitoring tasks that require focus over minutes or hours to catch infrequent events. Sustained alertness tasks with reaction time-dependent performance show increased reaction times, error rates, and instances of "sleep attacks," an event where attention lapses for tens of seconds mid-task causing a signal to be missed (Gunzelmann et al. 2009). If sustained attention is a major component for tasks on an interface, designers should consider the attentional requirements of the task and take advantage of tools like FAST (Fatigue Avoidance Scheduling Tool; Eddy and Hursh 2006) to plan work schedules that are compatible with the sleep patterns of the operators. For further information on sleep and circadian rhythms, it can be found in Wide Awake at 3:00 A.M.: By Choice Or By Chance? (1986) by R.M. Coleman. ## 3.3.2 Resuming Attention: Interruptions and Task-Switching Interruptions provide a major risk in disrupting the ability of operators to maintain their attention on a given task. Unanticipated breaks during the completion of a task have been shown to increase subjective workload and error rates, even for experienced professionals (e.g., Campoe and Giuliano 2017; DeMarco and Lister 1999). Campoe and Giuliano (2017) found that the errors when programming medical pumps occurred 7% more often when more than two interruptions occurred during the ≈5-min task. Designers should be aware of how interruptions, even when planned, can impair performance of operators. The overall framework for understanding task interruption can be divided into several phases. First, the worker will be completing some primary task. At some point prior to completing the primary task, the worker is exposed to a distraction signaling the need to complete a secondary task. The time between receiving the signal and initiating the secondary task is called the interruption lag. Next, the worker begins the secondary task. The time to complete the secondary task is called the interruption length. Upon concluding the secondary task, a period called the resumption lag occurs until the worker is able to resume the primary task (Trafton et al. 2013). This process can occur multiple times throughout the completion of a primary task. Distractions force the operator to lose their attention on one task, begin attending to a different task, and then transition back into attending to the original task. Each time the operator transfers their focus (in both directions), there will be a necessary "activation period" where the operator is working through the stages of situational awareness: perceiving the task features, forming a mental model of the situation, and fnally extending their mental model into likely future scenarios to guide action. This process takes time and leads to performance impairment. It is also a source of errors. Well-designed systems should attempt to alleviate the risks associated with interruptions to primary tasks. Systems engineers and designers can exhibit signifcant control over the design of the associated tasks. Although designers may be able to infuence operator training, it is more practical to design the system and tasks around a range of skill levels (when possible). The frst method for reducing the effects of interruptions on performance is simply removing them from the possible task structure. Even among experienced professionals working in high-stakes situations, the number of interruptions is directly correlated with an increased error rate, cognitive workload, and stress level (Campoe and Giuliano 2017). If interruptions cannot be limited, there are several ways to alleviate the performance impairment. First, designers can provide a preliminary warning signal that indicates an interruption is imminent (within the next 10 s). This allows operators to begin preparing to switch tasks (e.g., mentally noting a suitable stopping point) without the need to fully place their focus on the new task just yet. Trafton et al. (2003) informally describe the process that occurs after the warning signal as the operator answering two questions and storing the response in memory: "Now what was I doing?" "Now what am I about to do?" The answer to the frst question helps the operator identify the point from which to resume the primary task, thus reducing the resumption lag. The answer to the second question prompts the user to gradually begin attending to the interruption task, thus reducing the interruption lag. The same study demonstrated that providing a warning signal with 10-s notice for a distraction reduced the resumption lag by nearly 50% (8 s without warning vs. 4 s with a warning) for an unpracticed task. Although this effect diminished with repeated practice, this design guideline is particularly useful for infrequent tasks that may be minimally practiced. Besides offering a warning, designers can design interruptions that minimize the performance impairment. First, interruption length is a large predictor of the resumption lag. Working memory plays a signifcant role in managing attention. Long interruptions impair the ability to rehearse the previous task state and lead to an operator forgetting their place in the task. Designers can account for this by reducing the length of interruptions and preventing interruptions during high-stakes tasks (Campoe and Giuliano 2017). Interruptions that force the operator to change contexts also impair performance. Context change is a broad descriptor that may include changing locations, unexpected transitions from visual processing to verbal processing (e.g., talking to a coworker), or generally unexpected shifts in cognitive requirements (Marsh et al. 2006). So, when possible, allow the operator to fnish their current primary task step. This reduces the resumption lag for computer-based work, though this beneft appears to disappear for manual work (Campoe and Giuliano 2017). ## 3.3.3 Signal Thresholds and Habituation Visual input is naturally limited by the minimum stimulus strength that is detectable by the structures in the eye. The threshold that separates undetectable and detectable stimuli is called a detection threshold. For visual signals in the human eye, the threshold for light detection is approximately 100 quanta. The threshold corresponds to being able to detect a candle fame from 50 km on a clear dark night (Galanter 1962). The amount of change necessary to create detectable differences between stimuli is called a just noticeable difference (JND). We use JND to generally refer to a detectable difference as measured by the appropriate scale for the metric (e.g., decibels for sound). For example, let's say we ask a person to select the darker shade of orange between two similar, but different, orange color swatches. If the difference between the two is less than a single JND for the human visual system, then the person will perform no better than chance, even though a computer can instantly recognize a difference. A change in the interface display with less than one JND will have signals that are physiologically impossible to detect for the user. Thus, the signals and stimuli directed to the operator must be suffciently clear and distinct to be detected, and designers should avoid implementing visual features that communicate important changes through subtle differences. Although human vision can be very sensitive during the initial presentation of a stimulus, there is also a natural process of habituation that occurs during persistent detection of certain stimuli. As an operator becomes accustomed to a predictable, persistent visual stimulus, they lose the ability to perceive it without conscious effort; the stimulus becomes background to them. For example, people living next to train tracks stop noticing the trains. Though it is more common with simple stimuli, habituation can also occur with complex stimuli that require action (e.g., clicking a "confrm action" box for every action; Ritter et al. 2014). System designers already will be taking some steps towards accounting for these low-level issues during the design process. For example, system designers will often use particular visual characteristics such as fickering or fashing lights, changes in color, or motion to indicate that an operator's attention is needed. However, designers should use caution when deciding when to use alerting signals. When a system is working as intended, the designer should be aiming for signals that facilitate habituation, that is, the changes appear normal and do not call attention to themselves. However, once the system detects an alert of some kind, the design principles become inverted. Rather than facilitating habituation, designers should actively attempt to prevent habituation. ## 3.3.4 Speed-Accuracy Trade-off (Or How to Design for Acceptable Errors) There is a constant in human behavior represented by Fig. 3.7. This graph shows that behavior can be slow and careful with low errors, or rather fast and with higher errors. Operators will vary in what their curve looks like. Similar operators may be at different points on the same curve as well. To avoid the extremes, psychology studies often instruct subjects "to work as quickly and accurately as possible" to attempt to put subjects at some ideal center point along this curve. The center point allows fair comparisons between conditions in a study, but, typically, users will move along the curve to suit the task and situation. We note this speed-accuracy trade-off to designers so that when they are observing users, they realize that operators may be working at different points in the curve. For example, when typing drafts, we type quickly and use spell correction to clean up. When entering passwords, we type slowly because errors take time and force us to redo the whole task. ## 3.3.5 Summary of Attention Attention can be seen as the tasks and information that the operator is attending to or working with. There are consistencies and effects that arise from this process. To the extent that designers can understand the operator and their tasks, they have a role to facilitate the allocation of attention and to support its use. To summarize how designers can support operators' attention, we present a few design principles related to attention. #### Principle 3.6 Present information needed for comprehension directly Attention and working memory are limited; information shown to the operator should be processed and integrated as much as possible to reduce operator workload and support the system goals. Avoid giving operators extra work, particularly for tasks that can be automated or otherwise more effectively handled by the system. Methods for implementing this can range in complexity, but benefcial design choices will be structured around eliminating extraneous work for the operator. Simple examples might include reducing unnecessary mental math or just moving related information closer together. Eye movements take time, as do mouse movements. Making an interface easier to use with many small changes is important: milliseconds matter (Gray and Boehm-Davis 2000). Complex examples include totally redesigning a complicated display around a relatable design metaphor with a unifed representation of the information, as shown in Figs. 3.8 and 3.9. For example, consider a simple altimeter design. Pilots are often skilled operators with a lot of experience in their primary tasks. However, the human limits on attention and memory are always a factor. Designing to improve comprehension will reduce mental strain for experienced and inexperienced pilots alike. A pilot need not calculate the difference between assigned altitude and present altitude. Technology has advanced so that this can be calculated and displayed better than the initial dials. Simplify the task and use each system's strengths. The computer can handle simple mathematical calculations and could show the values using two lines separated by the deviation. The pilot can then identify any issues with altitude much more quickly with the visual process. Compare the two altimeters in Fig. 3.8. On Fig. 3.8a, the pilot must personally compute the difference, and direction of difference, between the present and Fig. 3.8 The interfaces for two different altimeters. The generic digital altimeter (a) requires the pilot to mentally compare their altitude to the set value while accounting for variables affecting the instrument accuracy. (b) The Garmin G500 simplifes this by including a spatial comparison between accurate barometric altitudes and clear representation of current altitude and ground level. (Used with permission, www.garmin.com) Fig. 3.9 The OZ display compared to a traditional cockpit. The traditional display (a) is an emulated display, and (b) shows the plane metaphor (top) used to develop the functional OZ cockpit display (bottom). Used with permission from Temme et al. 2003, pp. 75–77 assigned altitudes before responding accordingly. However, on Fig. 3.8b, the altitude difference is interpreted visually and is a much faster and less error-prone task. As another example that is more complex, consider Fig. 3.9 which shows the OZ display. It provides a redesign of an airplane's control panel around a direct implementation of an airplane metaphor. Flying with traditional airplane displays requires the pilot to mentally calculate their current fight relative to the limits based on the fight envelope (i.e., stable fight based on related parameters like airspeed, altitude, and orientation). This mental calculation is diffcult and cognitively taxing, particularly during times of high workload from adverse conditions such as fog or turbulence. When vision is impaired, pilots rely solely on instrument fight (IF) with no visual reference frame. This risky situation led Temme et al. (2003) to propose an interface titled "OZ" that portrays the key information as an integrated display built around a digital plane, shown in Fig. 3.9 (b, top). This display presents exactly what the pilot needs to know for the task: current aircraft performance compared to aircraft limits and optimal performance values. A comparison between old and new displays is shown in Fig. 3.9 (a and b, bottom). Although the OZ display in Fig. 3.9(b, bottom) appears complex to novice or unfamiliar users, it was designed to support common tasks that are familiar to pilots and is derived from the mental model used by the pilot during fight. The improvements from the new design were confrmed via tests showing that novice pilots using the OZ interface performed signifcantly better than novice pilots with the conventional display. With the OZ display, subjects with no fight experience immediately showed greater fight precision (for orientation and altitude) and reduced performance loss from turbulence than when using the typical display. After about 80 h of fight time with both displays, subjects attempted to perform a reading task while operating the plane. This task was essentially impossible with the conventional display, but subjects saw almost no loss in performance when using OZ. Similar designs could be created for control rooms, perhaps as a summary supporting task performance while retaining the raw data visible behind the summary display. #### Principle 3.7 Provide support for operators that may deal with interruptions. To summarize, to support operators so they can deal with interruptions: #### Principle 3.8 Consider the risks of stimulus habituation appropriately Even highly salient signals will become habituated with repeated presentation. Constant presentation of a signal leads to habituation, and thus reduced detection and attention by operators. Designers should create a hierarchy of signal salience to ensure the right signals get through to the operator. ## 3.4 Working Memory and Cognition Following the perception of information from the environment, the operator needs to use that information to make decisions and complete their work. Task-related information must be analyzed, manipulated, and transformed into useful information that can guide the actions taken by the operator. The operator must integrate their knowledge of the state of the world with their mental model of the task. For example, an operator sees that the temperature of some module is above the safe threshold and the battery is running low. The operator stores these facts in their working memory and then consults their long-term memory on how to respond to the issue. The response is then also added to working memory alongside the facts about the world state. The operator responds with the appropriate actions in the system, ensures the problem is fxed, and then discards the old information before moving onto their next task. Variations of this process occur many times throughout an operator's shift. These human memories do not work as well (at least under conventional views) as computer memory, so designers familiar with computers should be aware of the differences. Designers should particularly be aware of the differences because their own mental models of their own memories are likely to be particularly incorrect—if your memory fails, you are unlikely to be able to notice this! This section will describe how working memory and long-term memory affect operator performance. ## 3.4.1 Working Memory Often, the work performed in op centers requires operators to integrate snippets of information from various sources to come to a decision or understand the situation. This process of storing and manipulating that information occurs within the working memory of the operator. Working memory stores and manipulates information for near-term use (Ricker et al. 2010). Some tasks require multiple pieces of information to be analyzed and processed near-simultaneously; working memory enables people to handle this by offering a "scratch pad" for relevant information. Though particularly relevant during the performance of complex tasks, working memory is a foundational mediator for how each person interacts with the world. Working memory acts as a store for both internal events (i.e., recalling long-term memories) and external events (i.e., perceiving visual signals). In many ways, working memory is often analogized to be comparable to the RAM of a computer system, whereas long-term memory is like the ROM. The RAM, or working memory, allows rapid data access, effcient manipulation, and quick turnover between processes. The ROM, or long-term memory, provides a slower, semipermanent location for information storage and retrieval. The RAM–ROM analogy also applies to the limitations of working memory. While long-term memory does not appear to have a clear storage limit in humans, working memory is constrained by a capacity of only a few items—the most common general storage limit is about seven items plus or minus two items (Miller 1956). The seven-item limit is overly simplistic but provides a useful anchor for working memory capacity. Working memory capacity also varies across the population with greater working memory capacity being associated with better performance on cognitive tasks (Just and Carpenter 1992). The levels of abstraction and familiarity with the relevant concepts also have an effect; less abstract and more practiced tasks are easier to remember and use (Ritter et al. 2014, Ch. 5). The approximate limit for working memory capacity becomes even more complex due to processes such as chunking. Chunking refers to a mental process for grouping sets of individual information pieces into easily recognizable sets. For example, it will be easier to remember a sequence of items like "N S A F B I" (chunked as NSA, FBI) than "Q G Z T Y V" (not "chunkable" by most; Chalmers 2003; Ellis 1996). Chunking mechanisms can be leveraged by system designers to increase the practical working memory capacity of the users. Modern theories of memory suggest that working memory is built from specialized subsystems that differ based on their input: the "visuospatial sketch pad" for visual spatial information and the "phonological loop" for verbal information (Baddeley 2000). This distinction between verbal and visual working memory stores is important because these two systems can perform semi-independently without much interference (i.e., loss of performance) between them. When implemented successfully, this can allow someone to drive a car while listening to an audiobook with almost no loss of performance for the primary task (Granados et al. 2018). However, implementing this concept is not necessarily foolproof. When the secondary task requires too much mental effort (i.e., maintaining a conversation vs. passive listening), driving performance tends to be degraded to a noticeable degree (Strayer et al. 2003). Although multitasking is best avoided, making attempts to isolate the tasks to distinct working memory stores can provide some measure of risk reduction when it is impossible to eliminate the need for multiple tasks. For the designer, there are a few takeaway implications for design: ## 3.4.2 Cognitive Load Cognitive work is inherently taxing on our mental resources. We have previously discussed the impairment of cognition as it relates to attention, but higher-order processes are also affected. Throughout the performance of cognitive work within an op center, operators are presented with information that must be monitored and assessed and may need to be compared across time. These types of work are inherently diffcult, particularly when during long periods of performing the tasks. Cognitive load theory (CLT) describes how the various factors such as working memory load, personal stress, and task diffculty can provide an overall decrement on performance of cognitive work (Sweller 1988). Cognitive load theory provides a way to compare task diffculty (relative to the expertise of the user) across different task environments. Reducing cognitive load provides a broadly effective way to improve performance by freeing up working memory capacity for more important tasks like integrating information and learning. CLT currently lacks units and an objective way to measure it; however, we fnd CLT to be useful nonetheless because it provides a framework for comparing system design choices. A review of cognitive load's role in human–computer interaction design is provided by Hollender et al. (2010). Their review integrates CLT research into a useful framework for systems engineers. They posit three main types of cognitive load: intrinsic, extrinsic, and germane. Intrinsic cognitive load refers to the inherent complexity of the information being processed by the user. Comparing intrinsic load can only really be done by comparing two tasks rather than by providing a stand-alone value. For example, driving on an empty highway would likely provide less inherent complexity compared to driving on a busy city street. Extrinsic cognitive load refers to environmental and context-dependent factors that provide unnecessary contributions to task diffculty. Integrating spatially distant information from displays that are on opposite ends of the room will be inherently more diffcult than if the displays were side by side due to the required storage of the information in working memory between task steps. Finally, germane cognitive load refers to the benefcial cognitive work that improves task performance. Learning and practice of the skills and schema required to perform a task also require cognitive resources, in contrast to unhelpful portions of the overall cognitive load. All three types of load contribute to the overall working memory needs of any given task, and the ideal task will reduce the intrinsic and extrinsic load to provide more resources for the benefcial mechanisms that occur from germane cognitive load. Reducing the cognitive load of extraneous tasks can provide a consistently useful method for improving the performance of operators. A simple method for reducing cognitive load is by enforcing consistency across the layout, color scheme, and overall information presentation style for components of an individual system and across multiple systems (Chalmers 2003). Even experienced users that may switch between a Windows OS and Mac OS will know the feeling of attempting to use a Mac-only shortcut on a Windows machine (or vice versa). Many of the recommendations for reducing cognitive load can be succinctly described as follows: when possible, reduce the space and distance between codependent pieces of information. In some cases, it's a relatively simple process to fnd multiple solutions. Disparate information sources could be split across multiple displays to maximize information presentation, or alternatively, a single display could be trimmed of unnecessary information to bring the most important features onto a single, more effcient display (Brown et al. 2013). Other cases provide less clarity in determining the best practices for a given context. Providing redundancy in feature presentation can help reinforce certain information, but the additional features inherently increase the intrinsic cognitive load during interaction with the system (Grunwald and Corsbie-Massay 2006). Engineers and other stakeholders must use the risk-driven approach to make informed decisions; competing design recommendations are rarely weighted on easily comparable scales. Krug's (2005) approach provides further suggestions to reduce cognitive load that center around the titular message of the book: Don't Make Me Think. Krug argues that small design faws like unclear labels, confusing buttons, and unclear feedback introduce minor inconveniences that can add up and lead to a noticeable drop in overall system performance. Further ways to support operators and reduce cognitive load can involve shifting cognitively taxing tasks and information onto the system. This includes (a) reminding operators when tasks should begin; (b) reducing load by simplifying the number, length, and complexity of actions; and (c) automating tasks that can be automated, like how automobile turn signals automatically shut off after the steering wheel rotates back to straight. ## 3.4.3 Summary of Working Memory and Cognition Operators will be using their working memory on every task, but there are inherent limitations to capacity and processing power that need to be considered when designing the interface. Off-loading information to the system (when possible) reduces strain on working memory, as does simplifying or optimizing how information is displayed to leverage mechanisms like chunking to increase functional working memory capacity. By understanding the tasks and operators for their system, designers can identify ways to support operator performance through design choices. #### Principle 3.9 Reduce the cognitive resources used during multi-step tasks Operators' cognitive resources, including working memory and attention, are limited, and these limitations are made worse by fatigue, stress, and task diffculty. Simplifying the work will reduce workload and make errors less likely to occur. Simplifying tasks can be done in many ways depending on the specifc scenario. The common factor for all successful implementations of this guideline is a reduction in the amount of working memory, attention, or other cognitive resources needed to perform the task. For example, if an operator is alerted for a task that needs to be done in 30 min, the system should provide an additional reminder at the appropriate time rather than relying on the operator's memory. If a common task requires several steps to complete, provide an interactive task checklist that indicates the current state of the procedure—checklists are very helpful to support complex tasks. A simpler solution could be incorporating a window showing all inputs and outputs for the system with associated timestamps. ## 3.5 Summary The mechanisms that operators use while performing their work infuence how the work gets done, what errors are likely to occur, and how to design to support system success. This concept is common across other engineering felds. For an electrical engineer, the components that comprise electrical circuits infuence how circuits produce their outputs, what errors are likely to occur within the circuit, and how to design effective systems that require electrical circuits. The most salient mechanisms of operators that are relevant to improving the design of op centers are perception, attention, and working memory. These mechanisms interact, and good design will be based on a theory of how they are used by operators to perform their tasks based on the information presented to them in the interface. We include design principles to help with design. When these principles contradict themselves, which design principles and guidelines will inevitably do, the designers will have to resort to analysis of the tasks and their procedures, importance, and frequency to resolve the design trade-offs. There are also other mechanisms of operators, shown in Fig. 2.4, that will infuence performance in op centers. These mechanisms include motor output and other forms of perception. An overview of these mechanisms is available in Ritter et al. (2014). ## References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ## Chapter 4 Conclusion and Final Comments Abstract The foundational design philosophy of user-centered design (UCD) offers an ideal approach for systems engineers, programmers, designers, and any other stakeholder involved with the design of high-stakes systems with human operators. Furthermore, UCD, as presented here, is tailor-made to meet the unique needs of critical human–machine systems in systems like air traffc control towers, 911 call centers, or NASA's Mission Control Center. Whenever the operator is a missioncritical component of the system, stakeholders must be able to make informed decisions during the design process, and this book provides the tools necessary to make those decisions. ## 4.1 Introduction This book summarizes a process for designing and implementing op centers like the Water Detection System introduced in Chap. 1. As the work is performed, risks are assessed using a spiral development model that checks with stakeholders at each major phase, and adjusts the process based on the risks that can be perceived at that stage. The intermediate and fnal system can be assessed using simple usability tests as well as cognitive walkthroughs. The process uses shared representations of the operators, their tasks, and the context of the work. An example of these is provided in Appendix 1. These shared representations are used to design and create an op center. Appendix 1, with its subsections, provides an example set of documents for describing your users and their tasks in a way that is useful for design. Larger systems will need correspondingly larger and more complex descriptions, while smaller systems will typically need less. Systems only used by their developers might not need anything, but systems that are designed without these documents are designed informally and solely for their designer's use, not for the operators. As architects would discuss blueprints particularly before building a project, op center designers should expect to prepare and discuss these documents during design with other stakeholders, such as managers, future operators, and funders. These discussions can reduce misunderstandings, lead to supporting all the tasks for all stakeholders, defend designs, and help keep the relevant goals, missions, and tasks in mind when designing a system. Using these documents reduces risks (Pew & Mavor, 2007). Chapters 2 and 3 provide design principles that managers, designers, and implementers can be informed by. These stakeholders can also be informed by greater knowledge of the operators as a type of system component. Chapter 3 provides a short overview of the types of knowledge of operators that can help inform system design and implementation. Further sources for learning more are noted in each chapter. This book should also be seen as an initial review. There is more to know about how to support operators than is covered here. Appendix 2 provides pointers to further information on how to support operators in control rooms and to support the designers who create them. Appendix 3 aggregates the most important design principles that we have described in this book. The rest of this chapter briefy summarizes the book, offers areas of future work, and responds to the set of design questions presented at the end of Chap. 1. ## 4.2 The Need for User-Centered Design One of the diffculties with this approach will be investing the perceived additional time and effort to avoid the risks that this approach helps mitigate, ameliorate, or avoid. Typically, this approach takes additional effort, and organizations do not always see the risks until they arrive. There is evidence, however, that a mindful approach can overall reduce costs (Booher and Minninger 2005). A problem that remains then is to provide evidence that there are risks and that this approach helps reduce risks and their impact. Pew and Mavor (2007) call for examples to help motivate the different team members to appreciate how usability can infuence system performance. Table 4.1 notes a few examples. Support from management for this more engineering-based approach as well as further local examples could be useful to motivate implementers and technology designers to take operator tasks and their knowledge, skills, and abilities more seriously. Keeping a list of known risks and accidents related to the design domain could also be helpful in several ways. The particular risks to op centers' success may be diffcult to quantify and will often arise from unexpected events. It may be Table 4.1 Examples of usability problems leading to accidents (or extreme training or testing avoiding them). Further examples are available in Casey (1998) worthwhile for an organization to keep track of misses and near misses to accidents, as NASA does for air traffc control in the NASA Aviation Safety Reporting System (asrs.arc.nasa.gov/). ## 4.3 The Need for Better Shared Representations Another problem is the usability of the shared representations of users, tasks, and technology. Shared representations are documents about the design (e.g., types of users and tasks) that are shared across groups of stakeholders. The managers, designers, and implementers can come from different intellectual backgrounds, and have different assumptions. There is a need to translate some representations to "engineer speak," and perhaps in the other direction. There is a young literature on how to prepare knowledge about design aspects to share with other team members. This is a problem noted by Pew and Mavor (2007), where it is called shared representations, and work remains to make sure the shared representations are as usable as they can be. ## 4.4 Open Problems We can now revisit the questions in Table 1.4, presented here as Table 4.2. The responses are included in the table for convenience of reading and presentation. As the material in Table 4.2 notes, there remain open problems with applying this approach. The degree of detail required for the documents will vary across particular op centers, and across different technologies, and thus should be adjusted accordingly to the needs of the proposed system. The risks that arise in the use of particular op centers will vary with the domain that the op center is supporting. This approach does not guarantee a perfect or even a better system, but it overall reduces risk and the probability of system failures. ## 4.5 Ways to Learn More Designers of control rooms will need to know more about design and about operators than what is covered in this book. They will need to know more theory about design and human users, and they will need more details about the situations and operators and tasks that they are designing for. This appendix notes a few ways to learn more. These ways include reading, discussion, and formal and informal education. An hour a week of learning is not much in a week, but in a year, it can change how you think. #### Table 4.2 Questions addressed by this book 1. Which user interface features reduce user stress and improve and maintain level of performance? Situation awareness (SA) describes the operator's awareness of system state, and designs should support the cognitive processes used by operators to build up SA. Reducing cognitive load will reduce user stress, improve performance, support better SA, and help maintain performance over time. Cognitive load depends on multiple aspects of an interface, so matching the user, system, and tasks with the overall design will reduce stress and generally improve performance. Doing so is done by matching the user's capabilities with the interface 2. Which user interface design factors mitigate performance degradation (speed, accuracy) during the execution of detailed procedures for troubleshooting? The factors noted in answer 1 to start. Furthermore, designers should advocate for minimal task interruptions when possible, and support multitasking with helpful features when it is required. Developing and supporting SA in operators will also help reduce performance degradation by allowing high performance to be achieved while minimizing wasted cognitive resources High-throughput reaction times 3. Which features in fast and complex interfaces impair or enhance user reaction time and accuracy? The factors are detailed in Chaps. 2 and 3. Briefy, make perception of the task and task features quick, easy, and properly prioritized. Ensure that information presentation supports the mental model of the operator so they can have better SA. Improve the visual design and reduce cognitive load by reducing the type and number of substeps, and making the output able to be processed faster and more accurately by the operator 4. What are the reaction time and accuracy for a user to react to an alert and respond to the alert with the correct actions using the task user interface? What are the upper limits of the number and speed of alerts before performance degrades? We have ways to estimate the time to handle an alert. The keystroke-level model (Card et al. 1980, 1983) can be used to estimate response times. The upper limit must be based on an interface specifed in enough detail to make predictions. The feld does not have, to our knowledge, tools to fully compute the upper limit, because the limit would depend on many things that we don't yet have fully computational or algorithmic equations for 5. What are the reaction time and accuracy for a user to distinguish between levels of criticality using the task user interface? This time measure would depend on the perceptual display, the relatively frequency of signal and noise, and the payoffs between signal and noise. We do not know of an equation to compute this in general, but an equation could be created for fxed measures and validated empirically with operators 6. What are the effects of time-on-task (i.e., work shift length) on reaction time and accuracy for a user using the system? In general, with practice, reaction time goes down (Ritter et al. 2014; Chap. 5), but fatigue goes up. There are formulas to compute the general effect of fatigue (FAST; Hursh et al. 2004). They are validated but require some examination and understanding before use in a given situation. #### Table 4.2 (continued) Interface generalizability and individualized effectiveness 7. Which interface design elements vary and do not vary in effectiveness across various demographics? Design elements will vary based on previous experience with the design elements. The design elements would have to be specifed to fully answer this question. In general, designers should know the operators' tasks and make it easy to support each stage of SA by matching operators' capabilities with the interface 8. Which of the above questions are affected by age and prior education? All of these questions are affected by age and prior education. Typically, people become slower with age with raw response time, but this is typically not seen due to additional practice that contributes to lower response times as well as more knowledge which leads to better strategies and less search and problem-solving. Prior education that gives practice on the task or related tasks decreases time. Education that teaches useful theory will lead to better strategies that will in time, but perhaps not immediately, reduce response time. Further reviews are available in the cognitive and aging literature, and in the expertise literature, respectively ## 4.5.1 Readings to Learn More Designers wanting to learn more about design and operators can most easily read more. There are numerous books on how operators (as people) think and learn. A good book of this type is Anderson's Cognitive Psychology and Its Implications (2020). There are similar books for learning about perception (Sekuler and Blake 2005). Norman's (1988/2013) book helped start the area of human–computer interaction but does not provide a unifed theory of how to support design. It makes the case for paying attention to users and provides food for thought. As design moves in different directions, related books and textbooks can be found on broader topics such as the effects of emotions on our interactions with systems (Norman 2004). There are also books describing operators in terms that support design. Our favorite is Foundations for Designing User-Centered Systems: What System Designers Need to Know about People (Ritter et al. 2014), but textbooks by Wickens (e.g., Wickens et al. 2012) and Lewis and Rieman (1994) are also useful. If detailed knowledge about users is required, one can try to fnd the information in Boff and Lincoln's (1988) large compendium, but often the designer will be driven to reading more specialized papers, asking experts, running a study, or making an educated guess based on similar circumstances. Finally, the book Designing for situation awareness (Endsley et al. 2003) provides further useful advice. It will be familiar because we use it extensively in this book. We also recommend Sommerville's (2015) Software engineering (10th ed.), and particularly the chapters on reliability engineering (Chap. 11), systems engineering (Chap. 19), and systems of systems (Chap. 20). While not directly addressed in this book, Baxter and Sommerville's work on socio-technical systems brings a new perspective on the holistic design by integrating organizational change and system development into a unifed framework. There are also two fnal topics that we did not broach in this book: automation and the related topic of how operators use automation. Automation generally refers to the execution of some task that was formerly performed by a human. Eventually, some tasks will become fully automated with no future human interaction, at which point, these are simply machine tasks (Parasuraman and Riley 1997). Designers should be careful not to rush into automating tasks, particularly for complex tasks that will continue to rely on human input. Under perfect conditions, automation seems like an easy way to reduce the workload for your operators; however, when faced with the complications that reality brings, you can quickly run into issues. Operators use their trust in the automation to know how to use the automation and to then perform their tasks successfully with automation doing part of the task. Working with automation that is hard to calibrate can end up requiring more effort because the operator will need to monitor the automation to ensure success. Optimal performance can only be achieved when designers instill the proper amount of reliance and trust on the automated systems (Lee et al. 2004). The mental model of how the automation works and when it works should be accessible and easy to learn and easy to use. The process for automating tasks in complex systems is diffcult and outside the scope of this book, but we recommend reading Lee et al.'s (2004) article Trust in Automation: Designing for Appropriate Reliance and Parasuraman and Riley's (1997) article Humans and Automation: Use, Misuse, and Disuse if you wish to learn more. We also recommend reviewing NASA's Automation Interface Design Development project (https://techport.nasa.gov/view/23597). ## 4.5.2 Reading Groups One way to solidify knowledge from reading and to learn information not completely codifed is to participate in a reading group. Sometimes these groups appear as graduate courses. They can also be organized around a work group or, better, across work groups. They take time, but a group can help digest a book, and even the social loafers who do not read the material can learn something. It is also a way to build a shared theory of design in a workplace. ## 4.5.3 Continuing Education Finally, the most solid but expensive way to learn more is to take courses. Some will be available at local universities, and some are available online. Coursera and Lynda offer various courses that are related to these topics. ## References Anderson, J. R. (2020). Cognitive psychology and its implications (9th ed.). New York: Worth Publishers. Baxter, G. D., & Sommerville, I. (2011). Socio-technical systems: From design methods to systems engineering. Interacting with Computers, 23(1), 4–17. https://doi.org/10.1016/j. intcom.2010.07.003. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ## Appendices ## Appendix 1: Detailed Example Problem Space—The Water Detection System (WDS) Here is an example autonomous, asynchronous system that is used as a running example in this book. The goal of this fctitious use case is to enable readers to consider an example case that is typical of such op centers. The system description includes an overview, system architecture, key features, example day in the life (i.e., scenarios), typical issues, user types, and task analysis. Each of these could and should be expanded in more detail for real op centers. A set of these descriptions provides a solid basis for designing with the operator in mind. The initial draft of this system description has been created primarily by Mark Foster of L3Harris Technologies, and we have extended it over time. It is also a complete enough example of a remote autonomous, asynchronous system to be reused for other projects and courses. ## Overview SatCorp is an imaginary corporation that builds user interfaces for a unique class of command and control systems. These systems, while all unique, have many features that are consistent throughout their designs. The fctitious use case involves building a user interface to command and control a remote Water Detection System (WDS) that is based on an autonomous robot. It receives commands once per day and reports back at the same time. This WDS will be deployed to Mars in an attempt to detect pockets of water underneath the surface or traces of water in the soil on the surface. The WDS will take 5 years to develop and test before it is ready to deploy. Once ready, it will be sent to Mars as part of a larger manned mission. Due to space constraints on the manned vessel, the WDS will be disassembled before launch. It will be the responsibility of the team on this space mission to assemble the WDS, perform some initial checkout of the system, and ultimately deploy the WDS on the surface of Mars. During the assembly and checkout of the system, the team will command and control the system via a laptop with a local LAN connection to the WDS. The system checkout of the system is intended to exercise the different parts of the system to make sure they are still operational. Spare parts will be shipped with the system in case anything is damaged in transport. Once deployed on the surface of Mars, the WDS is expected to a have a 10-year mission where it is solely commanded and controlled by NASA's operation center. The operators in NASA's ops center are on duty 24/7. The WDS is only one of dozens of systems they monitor. Decision-making with regard to how the WDS is utilized comes from the scientists in the Program Offce, which funded the development of the WDS. It is the Program Offce's charter to fnd water sources in other locations throughout our solar system. This example (and associated material) ignores the communication delays with Mars because most op centers do not deal with such long time delays in communication media (although they will see delays in reports from other systems). #### System Architecture The WDS is comprised of several elements. These elements are listed with a brief description of each. Figure 1.4 (Chap. 1) diagrams the WDS and its connectivity to Earth. Main Control Element (MCE) The MCE acts as the brain in the feld. It is the responsibility of the MCE to facilitate commands from Earth and collect data and status to send back to Earth. More specifcally, when commands are sent to the WDS, the MCE oversees the execution of those commands. Commands that are scheduled for a future date will reside in the MCE until it is time to execute such commands. Commands for immediate execution will be executed upon receipt. Depending on the command type, the MCE is tasked with powering on the necessary elements and forwarding subcommands to those elements. All the while, the MCE is also constantly polling the other elements for status. In addition, the MCE provides storage for water analysis data from the Rock and Sand Exploration Element and Deep-Water Detection Element. When the WDS sends data home, it is the responsibility of the MCE to bundle element status and water analysis data, perform compression and encryption, and then forward that data when appropriate to the Communications Element. #### Communications Element (CE) The CE contains the antenna for communicating with Earth. This antenna is singleduplex and therefore can only receive or transmit at a given time. Due to this limitation, the antenna is by default in receive mode to receive commands from Earth. The team on Earth must command it into transmit mode to transmit data home. Typically, the team will schedule several transmit commands per day for updates from the WDS, but will only send commands to the WDS about once per week under standard operating procedures. #### Autonomous Navigation Element (ANE) The ANE controls the components of the WDS that are responsible for moving the WDS from one location to another. The ANE includes cameras for taking pictures of the terrain around it and has special image detection algorithms for identifying obstacles it must navigate around. The ANE can be commanded to move from point A to point B, and on its own determine the best route to get there, which may not be a straight line if obstacles are in the way. In addition, this element controls the drive motor, wheels, and steering functions. It also controls the emergency assist wheels and arms that enable it to get out of precarious physical situations. Rock and Sand Exploration Element (RSEE) The RSEE controls the shovel-like apparatuses that the WDS is equipped with. It also controls the cameras and sensors that are used to evaluate a segment of sand or rocks. Data recorded from this element are forwarded to the MCE for storage until being sent back to Earth for analysis. Deep-Water Detection Element (DWDE) The DWDE controls the drill and soil probe the WDS uses to search for water underneath the surface. When commanded to do so, the DWDE will drive the probe into the ground to gather water analysis data. In cases where the ground is too solid, the DWDE will remove the probe and use the drill to loosen the ground underneath the surface. After drilling, the probe is reinserted into the ground to continue gathering water analysis data. Like the RSEE, data recorded by this element are forwarded to the MCE for storage until being sent back to Earth for analysis. #### Power Generation Element (PGE) The PGE consists of solar panels and the system batteries. The PGE has a set of solar panels that are distributed around the WDS. These panels are used to generate power and charge the system batteries. The solar panels can rotate and tilt as needed to maximize sun exposure. The PGE is responsible for calculating the ideal rotation and attitude. #### Key Features of the WDS The following sections outline the key features that the user interface must accommodate. #### Status All six system architecture components listed above contain numerous status items that must be reported on a regular basis. Status items can range from environmental measures, such as pressure, temperature, and humidity, to element-specifc status such as current speed (mph or kph) for the ANE. One of the roles of the MCE is to periodically poll all the components for their latest status values. The MCE then stores all these values until the next opportunity to transmit data to Earth. All WDS components have redundant hardware with A and B sides for each element, so the MCE polls only the A or B side of a given component, depending on which side is currently booted. If the MCE attempts to poll a given component that is unresponsive, the MCE can power cycle that component or even switch sides of that component. This usually only occurs after some threshold of unresponsive polls. This threshold is confgurable. #### Event Logs Like reporting status, each component is recording an event log of the activities that component is executing. When a given component's log fle reaches a given threshold, that component will start a new log and transfer the old log to the MCE. The MCE will send all the logs home at the next opportunity to transmit data to Earth. The command and control GUI back on Earth will consolidate these logs into a single system log, but typically needs to flter out element-specifc details for display. #### Confguration Each element also maintains a set of confguration felds. For example, the MCE may be confgured to power cycle a given component after a certain number of unresponsive polls by the MCE. This value is confgurable under some scenarios because it may be appropriate to power cycle if three polls are unresponsive, whereas others may call for a wait period of ten polls without responding. In addition, whether to power cycle a component or power cycle a component and switch sides is another confgurable feature of the MCE. Another common confguration feld is which side of a component to use. The MCE holds a feld for each component, such that when commands are received from Earth, the MCE knows which side (A or B) of each component to power on to execute the commands. #### Commands Although commands are always sent to the MCE, each component supports a set of its own commands. For example, a Transmit command that is scheduled for 1 week from the current day would reside on the MCE's schedule for a week. Then, shortly before the Transmit command, the MCE would power on the CE and pass it the Transmit command and a bundle of data to transmit. At the scheduled time, the CE will execute the transmission of the data bundle back to Earth. #### Redundancy The WDS system will be deployed to Mars for a 10-year mission. During those 10 years, there will not be any maintenance missions, so every part of the WDS must have built in redundancy to assure the system can last 10 years. Except for the PGE's solar panels, every component has both an A and B side. For example, the MCE has two processor boards, one known as the A side and one known as the B side. The system only uses one at a time, but can be confgured to use either side. Furthermore, each side of a component has its own status. For example, the RSEE uses advanced moisture sensors to detect traces of water in the soil. In this case, the A side has a set of moisture sensors, and the B side has a completely different set of moisture sensors. Similarly, the network that connects all these components is also completely redundant, so there are A and B networks. #### Day in the Life A day in the life of the WDS is often unique. Table A1.1 shows a schedule for an example 24-hour period (24 hours per day, scaled from the Martian cycle). For the purpose of this use case, the Mars daylight hours will mirror those of Eastern Standard Time. Table A1.1 Example day for WDS (continued) Table A1.1 (continued) #### Example Issues The WDS is designed to autonomously handle issues that arise, but human interaction is required on a regular basis. Many of these tasks are simple maintenance and acknowledgement of warnings. For example, when batteries are low, the operator is required to acknowledge the low battery threshold. No action is required other than clearing the notifcation. Occasionally, however, the WDS will face an urgent problem that requires human input. These scenarios are rare, so the operator typically has limited training in how to address the issues. Here are some examples: Problem: The WDS is navigating in the crater and gets stuck. The operator from Earth must manually drive the WDS and control the ANE. The typical operator is not trained in this task, so the supervising manager must take control. The operators need to escalate the issue quickly because the WDS witnessed unexpected terrain. The mappings of Mars must be updated appropriately. Problem: Dust storm prevents batteries from charging. The MCE cannot task all the scheduled commands for the day. The CE alerts the NASA operators of the low battery status. The operator must re-task the day's commands because the ANE would use all the remaining power. This task is simple and can be completed by a novice employee but will require review by a supervisor. - If the WDS has a problem, it might take a few days for the engineers to remote in to fx the issue. Therefore, the overview screen will remain in a degraded (fault-shown) state. The problem arises when something else goes wrong on the system. For example, while at low power, a piece of equipment might overheat and be in danger of catching fre. The operators need to be alerted to this new degraded status and respond quickly. #### Stakeholder Analysis When designing a system, it is worthwhile keeping the stakeholders, the audience for the system, in mind (Boehm and Hansen 2001; Pew and Mavor 2007). Stakeholders for the WDS and other complex systems will have a similar structure as shown in Table A1.2. Direct users (i.e., operators), funders, and other stakeholders will each have their own requirements for the project. The stakeholders identifed for the WDS are described in the rest of this section. #### NASA 24/7 Operators Primary operators (or users) of the system are those who perform routine activity monitoring, respond to low-level alarms and events, and identify issues that require outside performance. They want a task that is within their knowledge, skills, and abilities and provides them with job satisfaction. Table A1.2 List of primary stakeholders and a brief overview of their role in the project The primary objectives of the 24/7 operators are to monitor the WDS for anomalies or issues and maintain communication with the WDS. It is the role of the operators to plan sets of transmit commands for the WDS system (which requires coordination with third-party communication systems) and send those commands to the WDS. Additionally, they must monitor the WDS interface to verify that the WDS has transmitted data to Earth when it is scheduled to. Upon receipt of this data, the operators perform a cursory review of the data to determine if there are any system issues that need to be addressed. In most cases, upon discovering a system issue, the operators will contact the Program Offce or Engineering Development team to troubleshoot the issue. Lastly, the operators are expected to respond to requests for information regarding the WDS. At any time, if the Program Offce or Engineering Development team needs some data points from the system, the operators should be able to retrieve that data for them. The risk of overall project failure due to operator abilities and needs is relatively more diffcult to specify due to the delay between operator feedback and interaction with the system. The most common sources of major failure will likely be due to unforeseen issues that are preventable by experienced (or lucky) operators who can react to the system beyond the pre-determined alarm and event conditions. For example, a system overheat event can lead to a positive feedback loop of further heating of other components that destroys key components. This could plausibly be detected by a perceptive operator, but system alert priorities might not directly reveal this as a critical issue until it is too late. A source of "minor" project failure could be through overall issues with design that lead to high error rates that increase project cost and reduce the perceived reliability of the system. Although an operator taking the wrong action (e.g., a command scheduling issue is frst reported to the system's development team before calling Program Offce scientists) is a relatively minor issue at frst, high error rates from operators increase the costs of the project and reduce the overall effectiveness of the operation center. The environment the operators work in is a command center that is staffed 24/7 with approximately 15 workstations. It is typically staffed with about 15 operators during the day and 10 operators at night. The primary environment is a "dim" room with desks in the center (i.e., not along the walls). The front wall, which all the desks face, is a wall of screens. The back wall, which no one faces, is a secondary wall of screens. Both walls of screens consist of multiplexed, disparate displays of 40–100 systems. Operators alternate in 12-hour shifts, with a day shift from 7 am to 7 pm and a night shift from 7 pm to 7 am. The night shift operators are typically former enlisted personnel, hence generally not college educated, and mostly in their early twenties. The day shift workers typically have a more advanced skill set than the night shift operators. The average age is greater compared to the night shift. The day shift operators tend to have more system knowledge and can handle slightly more advanced troubleshooting or analysis than the night shift. #### Operation/Command Center Supervisors Supervisors within the command center ensure operator performance and respond to high-level alarms and events upon notifcation by the primary operators. Like operators, the supervisors want job satisfaction and a task that is within their abilities. The supervisor's use of the system will share mostly the same set of risks as operators; risks to project failure will likely be the result of unforeseen issues that could be successfully caught with experienced or skilled workers. Supervisors act as the interface between the high-level management from NASA research scientists and the ground-level operators that directly interact with the op center systems. #### System Developers and Engineers The Engineering Development team is a cross-discipline team that has developed the WDS over the past 5 years. During the development phase, the WDS program consisted of hundreds of engineers; however, the program has now been reduced to essential personnel because the development is nearly ready for deployment. Most of the remaining personnel are software engineers, systems engineers, and integration/test engineers. This team's primary responsibility is to resolve bug tickets regarding the WDS software. This team is continually integrating and testing the latest software. Once a software release is ready, it will be loaded to the WDS, whether the WDS is still being used for training at NASA or if it has been deployed on Mars. In addition, any issues or anomalies with the system are investigated by the Engineering Development team in their development lab. The developers want mission success (as measured by other stakeholders), an easily programmed system, clear instructions, and to generally avoid "hard mental operations," leading to diffcult to program constructs, when possible. Developers will need to able to create the system within the constraints of the other stakeholders while also meeting their funding and time constraints. Besides these "common" risks that engineers should be familiar with, the other major risk of project failure facing developers is ensuring that all the needs of the system and users are met. The example of a major failure described under "operators" would partially be the fault of the developers (for not identifying the tasks and needs), the Program Offce scientists (for not providing an adequate list of tasks and needs), and possibly the op center supervisors, depending on the circumstances. However, the developers should make strides to gather this information or risk having their reputation be negatively affected (whether or not the failure is directly related to their decisions). The Engineering Development team works primarily in a large lab with the same equipment that will be or has been deployed on Mars. This enables the team to test the software releases and procedures before releasing updates. The team is available to address any issues that arise after deployment. The Program Offce scientists relay the issues that are presented by the NASA operators. Occasionally the Engineering team can interface directly with NASA to get their feedback on the WDS software, but this is usually limited. Therefore, the team must prioritize tasks based on Program Scientists' feedback. The Engineering team tests software updates with their mock hardware. #### NASA Program Offce Scientists The Program Offce scientists are highly educated individuals whose charter is to fnd water on Mars. This team is formally the customer for the Engineering Development team and, while colleagues of the operators, receives customer-like status when in the operation center. This team owns the decision-making on everything from design details to live mission judgment calls. They are the consumers of the water analysis data received from the WDS. They will use this data to generate reports for upper management at NASA and politicians. Their work heavily infuences the direction of our country's Space Program. This team decides where the WDS should navigate on Mars and when the WDS should attempt to gather more water analysis data. They need to be able to complete all necessary technical tasks (which are assumed to be known to the developers and engineers for the system). They also need to be able to interpret the data from the WDS, input and alter commands, and interact with the WDS via the same GUI as the operators that work within the operation center. Program Offce scientists should be able to provide an adequate set of requirements for the system or risk fnding out that their needs are unable to be met once the WDS arrives on Mars. The Program Offce scientists interface with the WDS via the same command and control GUI as the 24/7 operators. They frequent the operation center during business hours and especially around the time when transmit commands are scheduled with the WDS. While their primary expertise is in the science behind the water analysis data, they are fairly well versed with the WDS, as most of them have been a part of this program during the development of the WDS. Furthermore, most of them have experience working on similar systems deployed to other parts of the solar system. #### Project Funders and Other High-Level Stakeholders These are various individuals and organizations that oversee the project and provide funding for the work. They will be responsive to the assessments from the Program Offce scientists, explanations from the developers, and requirements from the supervisors within the operation center. However, they also have their needs and desires for the project. They may require design features based on a naïve understanding of the project's technical and scientifc needs. For example, they may prefer too great a consistency across projects (e.g., a common event log button across all systems), the use of incompatible software or hardware, or to prioritize a task (and interface elements) that does not correspond to other stakeholder needs. They also may provide necessary restrictions on work due to classifcation or other regulations that limit otherwise valuable sources of collaboration and feedback. They often want to have mission success with reduced resource costs. As the funders of the program, they will have their own expectations for project success. These expectations may differ from the assessments made by the Program Offce scientists, system developers, and other stakeholders. Many of the risks to system failure will come from lack of communication or miscommunication between the stakeholders. #### NASA Astronaut Install Team The astronaut install team is the last primary stakeholder for this project. They are responsible for assembling the WDS, conducting pre-deployment tests on the system, and launching it (thus releasing it from their responsibilities). This primarily provides technological requirements (e.g., the device must be able to be assembled with the resources available to the astronauts). Besides the technological requirements, they will need to be able to interact with the ground team to troubleshoot any issues or pass off the machine for remote troubleshooting via the operation center. The installation environment for the installers is obviously Mars. Therefore, their time is very limited as their mission is bounded by the resources (i.e., air, water, food, fuel) they have with them. Their energy levels are expected to be perpetually compromised after the extended time in space required to travel to Mars. Due to the annual meteor storm on the sector of Mars where the Program Offce desires the WDS to be deployed, the install team will not have communication with Earth during the installation. #### Summary and Lessons Each project will have multiple stakeholders. The list of relevant stakeholders is not simply limited to users that directly interact with the completed system or the implementers of the system. System success requires integration of the needs of the various stakeholders into a cohesive project plan that addresses their needs, capabilities, and abilities. This example system also has a wide range of stakeholders. Like other systems, there can be conficts and trade-offs between their goals. Table A1.3 Overview of the tasks for the NASA 24/7 operator for managing the WDS #### Task Analysis for 24/7 Operators The hierarchical task analysis developed for the NASA 24/7 operators provides a clear set of the most important tasks performed by the operators. The interface of a system should be designed to match the needs and capabilities of the stakeholders that are impacted by the interface. We focus on the 24/7 operators to provide a blueprint for the tasks that need to be accomplished using any interface designed for the WDS system. Table A1.3 gives an overview of the tasks described by the task analysis. Following the table is the detailed view of the tasks showing subtasks and other components. This task list would, through expansion, turn into an operation manual for any usability studies and a checklist for performing a cognitive walkthrough of the interfaces (Polson et al. 1992). The six tasks shown in Table A1.3 are an overview of the responsibilities for the operator of the WDS within an op center. Each task is decomposed into subtasks to identify the key steps and decisions taken by an operator while completing the task. Task 1: Periodic comprehensive review of WDS system Assumptions: WDS periodic update takes 300 ± 30 seconds. - (a) Find and check the WDS review schedule. - (b) Compare time for the scheduled review and the current time. - (i) If review is not necessary, end task. - (ii) If review is necessary, proceed to 2. - (a) If time before the next update is insuffcient, postpone until after next update end task. - (b) If time before the next update is greater than 3 min, proceed to 3. - (a) Complete the WDS periodic review checklist. - (b) Record the fndings of the checklist in the appropriate location and end task. Task 2: Repair or respond to any alarms following a WDS data update - (a) If alarm origin is PGE, proceed to 2-a-i. - (i) Check expected charge and determine if expected charge will bring battery above the acceptable threshold. - (ii) If charge will resolve alert, contact NASA Program Offce scientists and report overtasking of battery then return to 2. - (iii) If charging is low or nonexistent, contact WDS Development Team and report battery charging failure then return to 2. - (iv) If issue is unknown, contact op center supervisor and report unknown issue with PGE then return to 2. - (b) If WDS requires manual navigation control, proceed to 2-b-i. - (i) Contact op center supervisor and report WDS request for manual control. - (ii) Return to step 2. - (a) Determine cause of latching alert. - (i) If latching alert originated from WDS, proceed to 3-a-i-1. - 1. Find WDS element that sent the latching alert. - 2. Identify the command schedule fle used during the alarm. - 3. Report the command schedule fle, WDS element, and status data associated with the element to the NASA Program Offce scientists. - (ii) If latching alert did not originate from the WDS, proceed to 3-a-ii-1. - 1. Report the latching alarm origin and any other associated information to the op center supervisor. - (a) Identify special event priorities, if any, that have been requested by the NASA Program Offce scientists or op center supervisors. - (b) If new event notifcations match special event priorities, proceed to 4-b-i. - (i) If special event priorities include action plan for event occurrence, follow instructions from the action plan. - (ii) If no action plan is present, report event occurrence and associated data to the program that placed the special event priority. - (c) If no special priority events are found, dismiss all new events. 5. If all alarms, alerts, and events are processed, end task. Else, return to 2. Task 3: Ensure WDS transmits data to Earth per schedule and troubleshoot any delays Assumptions: The scheduled update timeframe includes a margin of error. - (a) If the update has not loaded and the update is not due, - (i) End task, and resume other duties. - (b) If update has loaded, ensure the next update time is shown and end task. - (c) If the update is not here and the update is due, check the margin of error for the update schedule. - (i) If within the margin of error, perform other duties until margin of error passes, and end task. - (ii) If update has not appeared after margin of error, continue to the next step. - (a) Check if the fle was received. - (i) Go to operation center event log. - (ii) Determine if a fle update event is found within update time frame within the op center event log. - 1. If fle not received, check for connectivity issues between satellite and operation center. - (a) If there are connectivity issues, call Comms team, inform of missing fle, and end task. - (b) If there are no connectivity issues, call WDS Development Team, inform of unknown cause of failed data upload, and end task. - 2. If fle was received, determine cause of failed update via event logs. - (a) Check operation center event logs and look for an application error. - (b) Check operation center event logs and look for an error processing fle. - (c) If either is found, call EIT, report the error, and end task. - (a) Find the WDS commands details module. - (b) Check the latest WDS command fle's end time. - (c) If the end time is more than 10 min away, end task. - (d) If end time is within 10 min, move on to 2. - (a) Verify that new command fle is ready for update. - (b) Schedule command fle update. - (a) Wait until next update from WDS. - (b) Check Comms event log for a successful command fle download during the last update cycle. - (c) If event is found within correct time window, end task. - (d) If no event is found, call op center supervisor, report fndings, and end task. Task 5: Responding to information requests regarding the WDS - (a) Isolate the requested event history. - (b) Transmit the requested event history to the NASA Program Offce scientists. - (a) Isolate the requested information for the NASA Program Offce scientists. - (b) Transmit the requested current status information to the NASA Program Offce scientists. Task 6: Respond to other events, alarms, and alerts that occur in non-WDS systems - (a) Request supervisor support if unsure of appropriate priority. - (a) Check for system changes. - (b) Identify stopping point for interrupted task. - (c) Complete interrupted WDS task. ## Appendix 2: Design Guidelines for Remote Autonomous Systems This appendix provides more detailed guidelines for desktop implementations of operation center interfaces than what has been covered previously in this book. The guidelines draw heavily on Apple's Human Interface Guidelines for desktop applications but are modifed to apply to the WDS system, its users and technology, and the users' tasks. These guidelines are annotated, modifed, and abridged to assist designers and engineers during the development of the applications and systems within operation centers. They are numbered and where appropriate sub-numbered. They are annotated according to four criteria: evidence level, testability, value added, and assessment for testing by the authors (Table A2.1). The criteria are represented after the guidelines in the following format: ## Example L guideline evel1 , , T V 1 N, o For this example, the format means that his guideline has some support from UCD and HCI experts (Level 1), could be easily tested for a given interface (T+), Table A2.1 Criteria defnitions for the design guidelines would not be much value to test (V1) for a given interface, and is not recommended for further testing by the authors (No). With regard to recommendation for further testing, we are not claiming that additional research is useless; rather, we just think that the benefts would not be worth the effort compared to other ways to spend limited .resources for design of a single interface. In the case of complex guidelines, like the frst guideline, we apply a general assessment of the claims made in the section without breaking down the fndings to every sub-statement. The various sub-statements might be guidelines or examples, and each statement might not have the same level of support. If only the high-level heading is rated on the criteria, please assume that the guidelines below that heading are a "set" that should be considered as a whole (e.g., Help and Tooltips under General User Interaction Guidelines). Otherwise, the high-level heading rating should be considered an overall assessment that is somewhat like an average of the ratings for individual guidelines. Finally, the support and evidence for the guidelines is provided in comments appended to the guidelines. A list of useful acronyms is described in Table A2.2. These will cover the majority of the evidence support, but some guidelines are also supported by links to full references to the research articles. Table A2.2 Common acronyms used throughout the guidelines and comments #### Introduction: Design Themes It is helpful for users to be able to anticipate design elements in an interface. It is useful, thus, for the elements to appear to be drawn using the same overall design framework with the same color palette, style and use of verbiage, style of tone, and word choice (e.g., word length, concreteness of words, use of articles, verb tense, and representational mapping). The same things should always appear as the same things, so differentiation can be reserved for useful, functional differences. Thus, conducting a design review after a multi-person team fnishes building an interface can be a useful method for improving the coherence of the design. A thorough design review will help pull the interface elements together and meld them into a coherent, intuitive whole that allows users to draw from a unifed set of task and context knowledge applicable across all of a company's systems. Design reviews can be made even more effective by implementing methods like heuristic evaluation by HCI experts on system design and cognitive walkthroughs to evaluate the system interactions. #### General User Interaction Guidelines Loading and Delays [Level 5], [T–], [V2], [No/Maybe] Operators want an application that acts on their commands and communicates how long processing will take. If your application presents blank or static content and does not provide feedback, people might think your app is frozen. - FDUCS §6.2.3: Feeling of Knowing and Confdence Judgments. Swift feedback helps users develop their knowledge for working with the system and avoid confusion. - LR §2.2.2 Stage 2 – Comprehension. Support comprehension by providing users with awareness of the system state. LR §2.2.2 Stage 2 – Comprehension. Support comprehension by providing users with awareness of the system state. - LR §2.2.2 Stage 2 – Comprehension. Support comprehension by providing users with awareness of the system state. Supporting Novice and Expert Users [Level 4], [T+], [V2], [Yes] Installation of op center systems may include up to 6 weeks of training to support new users; however, replacement workers may not receive that same support. These systems should accommodate experienced and novice users by providing in-system tools that enable learning of new tasks and reviewing procedures for uncommon or obscure tasks. 1. Establish a default confguration that's applicable to most or all operators. [Level 3], [T], [V3], [Yes] LR §2: Know yourusers,tasks. LR §3.1.5: Design to accommodate colorblindness. 2. Avoid unnecessary splash screens and instructions. Typically splash screens are fne for showing progress, but they are often just for show. If tutorials or intro sequences are necessary, provide a way to skip them. [Level 3], [T-], [V1], [No] FDUCS §11 & §12 on Task analysis; ADG. Splash screens can waste time, but also can be a source of feedback as the system loads. Splash screens can provide information at the expense of task effciency. - (a) Proactively look for times when people might be stuck. For obscure work and uncommon tasks, provide additional help in menus. - (b) Add help tags to system-specifc controls. - (c) Provide task-oriented documentation through a form of supplementary help documentation (either digitally or as a physical copy of a help document). - LR §2.2.2 Stage 2 Comprehension; LR §3.3 Working Memory and Cognition. Providing integrated help reduces cognitive load by reducing the amount of time spent searching for help and reducing the time and space between the issue and task completion. Including a help button would allow users to fnd help when needed and also provide a metric for which screens or tasks needed the most help. Testing could be done by comparing how usersrespond to in-system help, providing a physical help guide, and providing another option. - (a) Provide KSAs in menus to support learning. - (b) Base KSAs on typical Windows KSAs like ctrl-s for Save and ctrl-p for Print. - (c) Provide a full list of KSAs that can be viewed and/or printed out. GOMS, ADG, FDUCS. Clearly using KSAs would improve performance; however, outstanding questions include the value of KSAs for each task, time required to learn KSAs, and maybe others. Data Entry [Level 3.5], [T+], [V3], [Yes] Whether using a keyboard, mouse, or any other input mode, inputting information can be a tedious and sometimes error-prone process. When an app asks for lots of input before doing anything useful, people can get discouraged quickly. - LR §3.3. Working Memory and Cognition; FDUCS §10 Errors. Recall memory is slower, harder, and more error-prone than recognition memory. Even expert users are going to make errors at some point, so using recognition memory will reduce the number of errors and constrain errors to be within the known selection list. FDUCS §7.3.4 Scanning Displays and Menus. People tend to scan displays rather than deeply read them and the information should be presented in a scannable way that is sorted according to the operator's mental model. - LR §2.2.1 Stage 1 Perception; FDUCS §5.2.4.4 Priming; FDUCS §4.4.6 Pop-Out Effects; FDUCS §7.3 Reading; NN/g. Labels help users understand what they are looking at and prompt them to begin thinking about the relevant information needed for the task. Also, words are automatically processed for experienced readers so they will pop out upon being viewed by the user. Also, users read a word faster than naming an icon. - (a) Adjust text feld line breaks accordingly. By default, any text extending beyond the bounds of a text feld is clipped. A text feld, however, can be set to wrap text to a new line at the character or word level or to be truncated (indicated by an ellipsis) at the beginning, middle, or end. - (b) Consider using an expansion tooltip to show the full version of clipped or truncated text. An expansion tooltip behaves like a help tag and appears when the user places the pointer over the feld. ADG 6. Get information from the system whenever possible. Don't force users to provide information that can be gathered automatically or with the user's permission. [Level 4], [T], [V1], [No need] GOMS; CPM-GOMS; FDUCS §10 Errors: An Inherent part of human-system performance 7. Provide reasonable default values and prefll felds with most likely values when appropriate. [Level 3], [T+], [V2], [Maybe] GOMS; CPM-GOMS; ADG 8. Dynamically validate feld values rather than waiting until submission. This reduces the need to backtrack when data entry fails validation. [Level 3], [T+], [V3], [Yes] ADG; NN/g - (a) Displaying the input for percentages as a percentage or automatically presenting phone numbers in their standard format. - (b) Entries expecting long text should allow users to view the input with minimal scrolling (and thus less short-term memory usage). - (a) Always show commas for values above 1,000. - (b) Don't use "naked" decimal points: 0.5 is better than .5. - (c) Avoid showing trailing zeros for values that are always whole numbers: 1 is better than 1.0. - (d) When possible, build in automatic blocking of invalid numbers. - (e) Maximum stakes data entry felds can reduce risk of failure by using slightly larger decimal points and smaller font for numerals after the decimal. - (f) Batch long numbers in groups of three: 123 456 789 is better than 123456789. Help and Tooltips [Level 4], [T], [V2], [No/Maybe] Ideally, people can fgure out how to use your system without a guide. However, even in a highly intuitive interface, users sometimes need help learning advanced and secondary features. When called for, your program can offer assistance in the form of help tags and other forms of help documentation. Help tags allow you to provide temporary, context-sensitive help, whereas documentation allows you to provide a more thorough discussion of the topic. - (a) Try to limit tags to a maximum of 60 or 75 characters, depending on your system needs. - (b) Requiring more text to explain a feature may indicate that the interface is overly complicated. Keyboard Interactions [Level 4.5], [T+], [V3], [Yes] The keyboard is an essential input device for entering text, navigating, and initiating actions. Some users will prefer to use the keyboard for performing most or all tasks. #### GOMS, general wide support - (a) Full keyboard access mode lets users navigate and activate windows, menus, interface elements, and system features using the keyboard alone. - (b) Tab is an important command for switching between areas and felds. - (a) Unexpected shortcut design can easily confuse users, and it rarely makes sense to redefne a common shortcut. - (b) The WDS and similar systems could log commands to know which keyboard shortcuts and commands are most common. This would help improve keystroke accelerator generation. - (a) Maintain a consistent order using modifers and writing out commands with modifers. ## Providing User Feedback [Level 4], [T–], [V2] Feedback tells people what an app is doing and helps them understand the results of actions and what they can do next. ## FOK; CWT 1. Unobtrusively integrate status and other types of feedback into your interface. If a notifcation does not provide immediately actionable information, the operator should be able to continue their current task uninterrupted. [Level 4] [T], [V3], [Yes] #### LR §3.2.2 Interruptions 2. Avoid unnecessary alerts by carefully assessing whether new information is worth disrupting the operator's current task, so they can address the situation. If deemed important, ensure that the alert is disruptive enough to ensure the user responds. [Level 4] [T], [V3], [Yes] #### LR §3.2.2 Interruptions Badging or Icons as Updates [Level 3], [T], [V3], [Yes] The various systems in an op center can display small, meaningful icons to indicate new, noncritical information like events or minor alerts. FDUCS §7.3 How Users Read; Stroop on Automatic Processing of Words Notifcations [Level 3], [T+], [V3], [Yes] System notifcations provide timely and important information anytime. Notifcations may occur when a message arrives, an event occurs, new data is available, or the status of something has changed. FDUCS §7.3 How Users Read 3. If possible, ensure that responses prompted by the notifcation are not overly specifc or diffcult to accomplish once the notifcation is dismissed. [Level 3], [T–], [V2], [Maybe] LR §3.3 Working Memory and Cognition - (a) If the user is on the home page, then a notifcation about new events may be useful; if the user is already on the event log page, then displaying a pop-up will likely be annoying compared to other methods of informing the user of new event information. - (b) Critical events can implement cognitive counter-measures to capture the attention of the operator. Cognitive counter-measures are temporary, major changes to the interface intended to temporarily break their focus, so they will reorient onto the important task. For example, a low battery alert that occurs during manual control of an unmanned vehicle could clear the screen of all features and prominently display the low battery alert until cleared before resuming normal operation. This eliminates the risk of "tunnel vision" causing the signal to be missed. - (c) Critical events should use dual-coded alerts such as a visual and audio indicator or multiple visual indicators. - (a) Use the buttons to perform common, time-saving tasks. This will help reduce how often the operator needs to change views for simple tasks. Color [Level 4] [T+], [V2], [No/Maybe] Color is a great way to provide status information, give feedback in response to user actions, and help people visualize data. 1. Use color judiciously for communication. Limit the number of colors used for communication to fewer than fve. [Level 3], [T], [V2], [Maybe] ADG; LR §3.1.5 Principle 7 2. Provide adequate support for colorblind users. Colorblindness is common enough that, when possible, designers and engineers should ensure that the standard design supports colorblind users. [Level 4], [T+], [V2], [Maybe] LR §3.1 Perception 3. Color contrast should be between foreground and background colors should be at least 4.5:1, if not a higher contrast of 7:1. [Level 3], [T], [V1], [No] ADG 4. Test the application's color scheme under appropriate lighting conditions. A system used in a brightly-lit room will have different requirements than one used in a dark room. [Level 4], [T+], [V2], [No] LR §1.4 #### Visual Feature Index Most applications should be built using components from your preferred graphic design kit, such as Java Swing. This will provide a programming framework that defnes common interface elements. This framework lets applications achieve a consistent appearance across the system while at the same time offering a high level of customization. The following interface elements are a common set of fexible and familiar features that can provide a design framework for building nearly any system. Windows and Views #### Alerts An alert appears when the system or program needs to warn the user about an error condition or a potentially hazardous situation or consequence. A major alert within an application should be modal; once the alert is received, the program is locked into an "alert response" mode that requires user input regarding the alert before enabling any other actions. Minor alerts should be displayed differently than major alerts. - (a) Avoid using alerts to merely provide information. - (b) Users become annoyed at alerts and interruptions that don't provide actionable information. - (c) Avoid displaying alerts for common, undoable actions. Rieman, J., Young, R. M., & Howes, A. (1996). A dual-space model of iteratively deepening exploratory learning. International Journal of Human Computer Studies, 44(6), 743–775. https://doi.org/10.1006/ijhc.1996.0032 - (a) Consider phrasing a message as a question when the default action has some negative consequences (e.g., "Do you want to empty the trash?") - (b) Supplement alert messages with informative text. Use this space to elaborate on consequences, suggest solutions, and explain why the user should care. - (a) If the text and button titles are clear, there should be no need to explain the buttons. - (b) If guidance is needed, preserve capitalization when referencing buttons and don't enclose button titles in quotes. - (c) Give alert buttons succinct, logical titles. Best titles will use one- or twoword verb phrases that describe the result of clicking the button. Avoid using "yes and no" as the options. - (d) Label cancellation buttons appropriately. - (e) Include a Cancel button when there's a destructive button or action (e.g., delete fle). ADG 7. Ensure that the default button title refects the action the button performs. Avoid using OK unless the alert is purely informational. Specifc button titles like Erase, Convert, Clear, or Delete help users understand the action. [Level 3], [T–], [V1], [Maybe] CWT; ADG 8. Place buttons where people expect them. In general, the default (or most likely) button should be on the right. Cancel is usually on the left. [Level 2], [T], [V1], [No] ADG; Others 9. Consider offering time-saving keyboard shortcuts for all buttons. For example, Enter (or return) can a default "Accept" button for situations that are not high stakes. Clearly indicate defaults by using bold, underlined text (or another consistent graphic element) on the default choice. [Level 2], [T], [V2], [Maybe] ## Boxes [Level 2], [T–], [V1], [No] A box is a type of view that creates distinct, logical groupings of controls, text felds, and other interface elements. For example, a preferences window may include boxes that visually group related settings together. By default, a box has a border and a title, either of which can be disabled if it makes sense for your sub-display. The title, if displayed, can be positioned above (the default) or below the box. 1. Avoid nesting boxes. Nested boxes waste space and reduce the effectiveness of boxes overall for grouping information. ADG 2. Use sentence-style capitalization in box titles. Don't end box titles with a colon. APA guidelines; FDUCS §7.3 How Users Read ## Dialogs A dialog is a type of window that elicits a response from the user. Many dialogs like the Print dialog, for example—let people provide several responses at once. Dialogs are presented in three ways: document-modal, app-modal, and modeless. A document-modal dialog is attached to a document as a sheet and prevents the user from doing anything in the document until the dialog is dismissed. The user can still switch to other documents and apps. A Save dialog is an example of a documentmodal dialog. An app-modal dialog prevents the user from doing anything in the app until the dialog is dismissed. The user can still switch to other apps. An Open dialog is an example of an app-modal dialog. A modeless dialog is usually referred to as a panel. The user can continue interacting with documents and apps uninterrupted. The standard Find fle dialog is an example of a modeless dialog. #### Data Entry for Dialogs Dialogs are intended to be small, transient windows that don't require in-depth user interaction, so it's important to ensure that data entry is effcient. #### Layout - (a) Buttons in the bottom right of a dialog should dismiss the dialog. - (b) An action button, which initiates the dialog's primary action, should be farthest to the right. - (c) A cancel button should be to the immediate left of the action button. - (d) If a third button is needed, it should be to the left of the cancel button. - (e) If a help button is shown, it should be the furthest left button. - (a) For example, Don't Save should be far enough away from Save to ensure accidents are rare. - (b) Destructive buttons should require intentional effort. - (c) Ideally, 24 points of separation is best. ## Dialog Dismissal ## Outline View [Level 3], [T+], [V3], [Yes/Maybe] An outline view presents hierarchical data—like folders and the items they contain cleanly and effciently in a scrolling list of cells that are organized into columns and rows. At minimum, an outline view includes one column that contains the primary hierarchical data: parent containers and their children. Subsequent columns may be added, as needed, to display additional attributes that supplement the primary data. Event logs could be presented in outline view as an alternative to the typical table view. - Bakke, E., Karger, D. R., & Miller, R. C. (2013). Automatic layout of structured hierarchical reports. IEEE Transactions on Visualization and Computer Graphics, 19(12), 2586–2595. https://doi.org/10.1109/TVCG.2013.137. The outline view is just one way to present data. There could be valuable testing done on how best to present complex sets of events from the WDS and other systems based on the mental model of the user. ADG 3. If deemed appropriate, operators should be able to click column headings to sort an outline view. Clicking again should sort the column in the reverse order of the initial click. [Level 2], [T], [V1], [No] ADG ## Panels A panel is an auxiliary window containing controls, options, or information related to the active document or selection. A panel appears less prominent than a main window and can behave like a normal window or be confgured to foat above other open windows—even modal windows. Panels can also adopt a darker, translucent appearance when the UX calls for it. - (a) HUD panels are translucent and typically have a darkened background. - (b) Use simple controls and interactions for HUD panels. Avoid making the user type, for example. - (c) Keep HUD panels fairly plain with minimal color and other distracting features. ## Popover [Level 2], [T–], [V1], [No] A popover is a view that appears above other content on screen when you click or mouse-over a control or view. Popovers typically integrate an arrow pointing to its origin. Popovers can close in response to a user interaction (transient behavior), in response to a user's interaction with the view or element from which the popover emerged (semi-transient behavior). A popover can also be made detachable. A detachable popover becomes a separate window when dragged by the user, allowing it to remain visible on screen while the user interacts with other content. ## ADG ## NN/g Scroll View [Level 3], [T+], [V2], Yes/Maybe A scroll view lets people browse content (e.g., a large event log) that is larger than the view's visible area. A scroll view itself has no appearance, but can display horizontal and vertical scroll bars, each of which consists of a track containing a draggable control known as a knob. The height/width of a knob refects the quantity of scrollable content. - GOMS; Bakke, E., Karger, D. R., & Miller, R. C. (2013). Automatic layout of structured hierarchical reports. IEEE Transactions on Visualization and Computer Graphics, 19(12), 2586–2595. https://doi.org/10.1109/ TVCG.2013.137. Event logs are complex sets of data that need searched by users, and determining the best way to present them could be valuable. - GOMS; LR §3.3 Working Memory and Cognition. Scrolling requires the user to store more information in working memory rather than "maintaining" that information on the screen. ## Split View A split view manages the presentation of two or more panes of content. Each pane can contain any variety of elements, including buttons, tables, column views, text felds, and even other split views. The panes of a split view can be arranged horizontally or vertically and are separated by a divider that can typically be dragged to resize the panes. Each pane can have a minimum and maximum size, which affects how much it can be resized. Many apps let the user hide specifc panes on request. ADG ## Tab Views [Level 3], [T], [V2], [No/Maybe] A tab view presents multiple mutually exclusive panes of content in the same area. A tab view includes a tabbed control (which is similar in appearance to a segmented control) and a content area. Each segment of a tabbed control is known as a tab, and clicking a tab displays its corresponding pane in the content area. Although the amount of content can vary from pane to pane, switching tabs doesn't change the overall size of the tab view or its parent window. ADG 3. Controls within a pane using tab view should only affect content within that tab. [Level 2], [T], [V1], [No need] ADG; CWT ADG; GOMS Menus [Level 3], [T+], [V2], [Yes/Maybe] A menu presents a list of items–commands, attributes, or states–from which a user can choose. An item within a menu is known as a menu item and may be confgured to initiate an action, toggle a state on or off, or display a submenu of additional menu items when selected or in response to an associated keyboard shortcut. Menus can also include separators, and menu items can contain icons and symbols, like checkmarks. ## CWT; GOMS; ADG 1. Use title-style capitalization for all text. [Level 2], [T], [V1], [No need] APA Guidelines; ADG 2. Ensure menu titles are intuitive so users will anticipate the types of items the menu contains. [Level 4], [T], [V2], [Maybe] ADG; NN/g; CWT; Information scent research 3. Keep menus enabled, even when menu items are unavailable. [Level 3], [T], [V2], [Maybe] ADG; CWT; Mendel, J., & Pak, R. (2009). The effect of interface consistency and cognitive load on user performance in an information search task. Proceedings of the Human Factors and Ergonomics Society Annual Meeting, 53(22), 1684–1688. https://doi.org/10.1177/154193120905302206 FDUCS §7.3 How Users Read; ADG 5. Only use text for menu items. Icons are confusing and unnecessary. [Level 3], [T], [V1], [Maybe] FDUCS §7.3 How Users Read; Ghayas, S., Sulaiman, S., Khan, M., & Jaafar, J. (2013). The effects of icon characteristics on users' perception. In International Visual Informatics Conference (pp. 652–663). 6. Ensure the menu titles and text make sense according to their function. [Level 2], [T], [V2], [No] ADG; NN/g - (a) If necessary to include a submenu, only have a single additional level to the menu. - (b) Avoid having more than fve items in a submenu. - (c) Only consolidate related menu items into submenus. For example, Sort By Name, Sort By Date, and Sort By Length could be merged into a single command Sort By with a submenu for Date, Name, and Length. FDUCS §7.3.4; GOMS; St. Amant, R., Horton, T. E., & Ritter, F. E. (2004). Model-based evaluation of cell phone menu interaction. Proceedings of the SIGCHI Conference on Human Factors in Computing Systems, 6(1), 343–350. https://doi.org/10.1145/985692.985736 GOMS; LR §3.3 Working Memory and Cognition. Scrolling requires the user to store more information in working memory rather than "maintaining" that information on the screen. 11. If icons are necessary for your menus (such as for a toggled setting), use a standard, limited set of clear symbols like a checkmark. [Level 2], [T], [V2], [Maybe] ADG #### Contextual Menus A contextual menu, or shortcut menu, gives people access to frequently used commands related to the current context. Contextual menus are typically brought up by using a right-click on the item. Contextual menus often provide a limited set of useful actions that are frequently used in a particular situation. ## Buttons ## Checkbox [Level 3], [T], [V1], [No] A checkbox is a type of button that lets the user choose between two opposite states, actions, or values. A selected checkbox is considered on when it contains a checkmark and off when it's empty. A checkbox is almost always followed by a title unless it appears in a checklist. ## ADG; Tufte ## Gradient Button A gradient button initiates an immediate action related to a view, such as adding or removing table rows. Gradient buttons contain icons—not text—and can be confgured to behave as push buttons, toggles, or pop-up buttons. They usually reside in close proximity to (either within or beneath) their associated view. #### ADG ## Help Button [Level 3], [T+], [V3], [Yes] A help button appears within a view and opens application-specifc help documentation when clicked. All help buttons are circular, consistently sized buttons that contain a question mark icon. - (a) Dialog with dismissal buttons (e.g., OK and Cancel): lower-left corner aligned with dismissal buttons. - (b) Dialog without dismissal buttons: lower-left or lower-right corner. - (c) Preference window or pane: lower-left or lower-right corner. #### Push Buttons [Level 2.5], [T–], [V1], [No] A push button appears within a view and initiates an instantaneous app-specifc action, such as printing a document or deleting a fle. Push buttons contain text—not icons—and often open a separate window, dialog, or app so the user can complete a task. #### ADG; NN/g #### Radio Button [Level 2.5], [T–], [V1], [No] A radio button is a small, circular button followed by a title. Typically presented in groups of two to fve, radio buttons provide the user a set of related but mutually exclusive choices. A radio button's state is either on (a flled circle) or off (an empty circle). A radio button can also permit a mixed state (a circle containing a dash) that's partially on and partially off. However, it's better to use checkboxes when your app requires a mixed state. #### ADG; NN/g; GOMS; General support from work on visual scanning 7. In almost every case, pre-select a radio button to indicate the default selection. Default buttons reduce confusion and can allow engineers to imply the best course of action to the user. ## Fields and Labels ## Combo Box A combo box combines a text feld with a pull-down button in a single control. The user can enter a custom value into the feld or click the button to choose from a list of predefned values. When the user enters a custom value, it's not added to the list of choices. ## Labels [Level 3.5], [T], [V2], [Yes] A label is a static text feld that describes an onscreen interface element or provides a short message. Although people can't edit labels, they can sometimes copy label contents. - (a) Typically labels for controls should end with a colon. An exception to this rule is when the label and control form a complete sentence. - (b) Use system-provided, standardized label colors to communicate relative importance. ADG; GOMS; CWT; LR §3.3 Working Memory and Cognition 3. Labels and other text must use a consistent vocabulary, syntax, and grammar. Even minor changes can have a negative impact on the mental model and understanding of the user. [Level 4], [T+], [V3], [Yes] NN/g; https://www.nngroup.com/articles/visual-indicators-differentiators/ #### Search Field [Level 3], [T+], [V3], [Yes] A search feld is a style of text feld optimized for performing text-based searches in a large collection of values. Many windows include a search feld in the toolbar, but a search feld can also be displayed in the body area of a window. A search feld typically displays a magnifying glass icon and can also include placeholder text and a cancellation button. ADG; NN/G; CWT; Others 1. Ensure search felds have a distinct look that users can instantly recognize and distinguish from other similar features like text felds. [Level 3], [T], [V1], [Maybe] ADG; Mendel, J., & Pak, R. (2009). The effect of interface consistency and cognitive load on user performance in an information search task. Proceedings of the Human Factors and Ergonomics Society Annual Meeting, 53(22), 1684–1688. https://doi.org/10.1177/154193120905302206 ADG; NN/g. See https://www.nngroup.com/articles/suggested-employeesearch/. 3. Scope bars, a type of toolbar for fltering searches, will help users trim down unnecessary information during searches that may bring up large amounts of data. [Level 2.5], [T], [V2], [Maybe] ADG; NN/g; CWT (a) Plan scope bar functions around the tasks. Searching documentation for a page might not need detailed search flters; however, searching an event log with thousands of entries may require users to input multiple flters. ADG; LR: §2.2.1 Stage 1 – Perception 5. Filtering for date ranges should have multiple input methods like text view and calendar view. [Level 2], [T+], [V2], [Maybe] ADG; NN/g 6. Ensure that date formats are clear. #### Text/Character Field [Level 3], [T], [V2], [Yes] A text feld is a rectangular area in which the user enters or edits one or more lines of text. A text feld can contain plain or styled text. Text felds are the base category for search felds, labels, and other related features. #### ADG; NN/g 1. When providing a user-provided data entry feld, use a clear label with useful hints close by to communicate the purpose of the text feld. [Level 2], [T], [V1], [No] 2. Perform feld validation after the user fnishes typing into the feld. Don't wait until the user tries to submit the data. [Level 3], [T], [V2], [Yes] ADG; NN/g; Others. The value of this is dependent on what is being typed. For numerical entry, this is more important. - LR §3.3 Working Memory and Cognition. Being unable to view the full text feld forces operators to store information within working memory rather than simply view it if they want the full picture. LR §3.3 Working Memory and Cognition. Disappearing placeholder text can strain working memory, particularly when distracted. Mendel, J., & Pak, R. (2009). The effect of interface consistency and cognitive load on user performance in an information search task. Proceedings of the Human Factors and Ergonomics Society Annual Meeting, 53(22), 1684–1688. https://doi.org/10.1177/154193120905302206 - FOK; Mendel, J., & Pak, R. (2009). The effect of interface consistency and cognitive load on user performance in an information search task. Proceedings of the Human Factors and Ergonomics Society Annual Meeting, 53(22), 1684–1688. https://doi.org/10.1177/154193120905302206 - ADG; Consistency and cognitive load on user performance in an information search task. Proceedings of the Human Factors and Ergonomics Society Annual Meeting, 53(22), 1684–1688. https://doi. org/10.1177/154193120905302206 #### Date/Time Picker [Level 3], [T], [V3], [Yes] A date picker lets the user choose a date, a time, a date and time, or a range of dates. Date and time can be presented in a textual format using text felds, as a graphical format using a calendar view and/or clock view, or as a display showing both at once. #### KLM; CWT ## Segmented Control A segmented control is a horizontal set of two or more segments, each of which functions as a button, and is usually confgured as a toggle. Segmented controls provide closely related choices that affect an object, state, or view. Like buttons, segments can contain text or icons. A segmented control can enable single choice or multiple choices. ## ADG; LR §2.2.2 Stage 2 – Comprehension ## Level Indicators [Level 3.5], [T+], [V3], [Yes] A level indicator graphically represents of a specifc value within a range of numeric values. It is similar to a slider in purpose, but is more visual and doesn't contain a distinct control for selecting a value—clicking and dragging across the level indicator itself to select a value is supported, however. A level indicator can also include tick marks, making it easy for the user to pinpoint a specifc value in the range. A capacity indicator illustrates the current level in relation to a fnite capacity. Capacity indicators are often used when communicating factors like disk and battery usage. #### Progress Indicators [Level 4], [T], [V2], [No] Don't make people sit around staring at a static screen waiting for your app to load content or perform lengthy data processing operations. Use progress indicators to let people know your app hasn't stalled and to give them some idea of how long they'll be waiting. There are two general kinds of progress indicators: bar indicators and spinning indicators. Bar indicators (or progress bars) use a horizontal bar that flls from left to right to show the progress of some action. Spinning indicators use a circular form to show progress through flling the circle as progress continues. - 1. Progress indicators should only be shown within a view, not in window frame areas like toolbars and status bars. 12. Spinning progress indicators typically won't need labels. #### Some Parting Advice for Designers #### Guidelines Will Not Cover All Decisions Guidelines cannot cover all instances. There may be edge cases or places where unexpected questions arise about design: for example, another item to add, another task to add, or a different type of screen or user. The guidelines might also contradict themselves, which will require theory or an experiment to resolve. The implementer will often be asked to make short-term, rapid design decisions without the requisite time or resources to properly analyze the situation. For example, a customer may determine that the power module requires a view showing power over time in addition to the current power level. Should the power-over-time view be shown in addition to the current power level or merged into a single view? Should the powerover-time view change the line's color to show low-power alerts or use a horizontal threshold line instead? Providing implementers, designers, and engineers with additional training will allow them to make good design decisions throughout the design process. Even design guidance will not always provide enough information to implement a system. Better systems are built when the implementer is at least sympathetic to and perhaps even has studied a bit about the domain they are implementing. Architects who understand how buildings are built provide better, easier to build buildings, and architectural engineers build better buildings if they have studied architecture. The same holds true for systems engineers, UX designers, and the various other groups that contribute to creating the systems that reside in op centers. Engineers who understand their users and other stakeholders will build better interfaces. #### Study the User Thus, interface implementers should study the user slightly to be prepared for when, explicitly or implicitly, decisions must be made while implementing the interface. This might take 10–25 hours a year. #### Study How to Design Interface design and implementation is a process and procedural skill like any engineering discipline, similar to writing code, writing English, or even medical practice. Professionals in this area should get continuing education in the process of design. This might take 10–25 hours a year. ## Appendix 3: All Design Principles Described in This Book This appendix lists all the design principles that are covered in the book. Some design principles are grouped and presented as a table (Table A3.1). Table A3.1 Aggregated list of design principles covered by this book ## References ## Author Index #### A Acromite, M., 54 Altmann, E.M., 30 Anderson, J.R., 31, 67 Angell, L.S., 26 AuBuchon, A.M., 56 #### B Baddeley, A.D., 33, 57 Balkin, T.J., 66 Banbury, S., 24 Baxter, G.D., 8, 10, 22, 31, 67 Belenky, G., 66 Besnard, D., 31 Bias, R.G., 17 Biddle, R.L., 58 Blackmon, M.H., 12 Blake, R., 67 Boehm, B., 6 Boehm-Davis, D.A., 53 Boff, K.R., 26, 67 Bolkhovsky, J.B., 49 Bolstad, C.A., 26, 30, 31 Booher, H.R., 64 Boring, R.L., 44, 45 Bovair, S., 31 Brock, D.P., 30 Brown, J.M., 58 Brown, J.S., 40 #### C Cairns, P., 11, 93 Campoe, K.R., 49–51 Card, S.K., 10, 12, 66, 88 Carpenter, P.A., 56 Casey, S.M., 64 Chalmers, P.A., 56, 58 Chang, D., 41, 43 Chilton, E., 27 Chipman, S.F., 64 Chon, K.H., 49 Churchill, E.F., 8, 10, 22, 67 Cook, G.I., 50 Corsbie-Massay, C., 58 Cowan, N., 56 Cox, A.L., 11 Cox, D., 10 Cuevas, H., 26 #### D DeMarco, T., 49 Deneke, M., 58 Dinges, D.F., 49 Dooley, L., 41 Dow, S., 18 Drews, F.A., 57 #### E Eddy, D.R., 49 Egeth, H.E., 41 Ellis, N.C., 56 Endsley, M.R., 2, 22, 24, 26, 27, 29–31, 67 Engle, R.W., 48 Enns, J., 40, 41 Epling, S.L., 49 © The Author(s) 2021 119 J. D. Oury, F. E. Ritter, Building Better Interfaces for Remote Autonomous Systems, Human–Computer Interaction Series, https://doi.org/10.1007/978-3-030-47775-2 #### F Findlay, J.M., 40 #### G Galanter, E., 51 Garner, W.R., 39 Gilchrist, I.D., 40 Giuliano, K.K., 49–51 Gluck, K.A., 49 Gorton, T., 24 Granados, J., 57 Gray, W.D., 53, 88 Greathead, D., 31 Greenspan, S.L., 58 Gross, J.B., 49 Grunwald, T., 58 Gunzelmann, G., 49 #### H Hansen, W., 6, 78 Harrison, A.M., 49, 50 He, J., 57 Healey, C., 40, 41 Helton, W.S., 48, 49 Hiatt, L.M., 50 Hick, W.E., 18 Hicks, J.L., 50 Hofmann, C., 58 Hollands, J.G., 67 Hollender, N., 58 Hopper, M., 57 Hursh, S.R., 49 Hyman, R., 18 #### I Irwin, D.E., 40 #### J Johnson, M.L., 66 Johnston, W.A., 57 Jones, D.G., 2, 22, 27, 29–31 Just, M.A., 56 #### K Khemlani, S., 49 Kieras, D.E., 31, 64 Kitajima, M., 12 Kosslyn, S.M., 29 Krug, S., 30, 58 #### L Lewis, C., 11, 12, 17, 67, 88 Lincoln, J.E., 26, 67 Lister, T., 49 #### M Marsh, R.L., 50 Mavor, A.S., 6–11, 17, 64, 65, 78 Mayhew, D.J., 17 Miller, G.A., 56 Minninger, J., 64 Mintz, F.E., 30 Moore, C.M., 41 Moran, T.P., 10, 12, 66 Moray, N., 31 #### N Newell, A., 10, 12, 66 Norman, D.A., 67 #### O Oury, J.D., 32 #### P Pew, R.W., 6–11, 17, 64, 65, 78 Polson, P.G., 11, 12, 83, 88 #### Q Qin, M., 49 #### R Redmond, D.P., 66 Ricker, T.J., 56 Rieman, J., 11, 17, 67, 88 Riley, J.M., 30, 68 Ritter, F.E., 9, 10, 17, 30, 31, 39, 42, 46, 48, 51, 56, 60, 66, 67, 88 Russell, P.N., 48 #### S Schmitz, B., 58 Schrock, J.C., 48 Schultz, A., 49 Sekuler, R., 67 Selcon, S., 24 Sommerville, I., 67 Still, D.L., 54 Strayer, D.L., 57 Stroop, J.R., 38 Sun, J., 40 Sweller, J., 57 #### T Tamborello, F., 49 Tatlock, K., 24 Tehranchi, F., 31, 32 Temme, L.A., 54 Thorne, D.R., 66 Trafton, J.G., 30, 49, 50 Tufte, E.R., 29, 31, 100 Tuovinen, J.E., 41 U Ulrich, T.A., 44, 45 Unsworth, N., 48 #### W Wang-Costello, J., 26 Warm, J.S., 48 Wharton, C., 11 Wickens, C.D., 67 #### Z Zeki ć, K., 3 ## Subject Index #### A A/B experiment, 11 See also Study ACT-R, 31, 32 See also Cognitive model Aesthetics, see User-experience design (UX) Age, 19, 67, 79 Aircraft, 31, 54 Air disasters, 4 Air traffc controller(s) and control (ATC), 31 Alarms, 27, 28, 39, 43, 45, 47, 78–80, 83, 84, 86, 111 Alerts, 13, 16, 19, 28, 31, 47, 51, 66, 77, 79, 83, 84, 86, 95–99, 101, 113, 115 Altimeters, 53 Apple Human Interface Design Guidelines, 85 Astronauts, 1, 16, 78, 82 Attention attentional failure, 4, 27 attentional vigilance, 48 goal-directed attention, 40 Audition/auditory, 38 Automation, 67, 68, 97 Aviation disaster, see Air disaster Automobile, 23, 25, 59, see Car interfaces, driving #### B Badging, 95–96 Buttons, 58, 81, 89, 91, 96, 97, 99–101, 103, 104, 107–110, 113 #### C Car interfaces, 23, 24, 26, 28 Checkboxes, 107, 108 Chunking, 56, 57, 59 Circadian rhythms, 49 CMN-GOMS, see Keystroke-level model, GOMS Cognition, 27–30, 32, 37–60, 90, 91, 96, 103, 106, 109, 111, 116 Cognitive, 4, 8, 11, 12, 22, 25, 26, 31–33, 37, 39, 40, 48, 50, 56–59, 63, 66, 67, 88–90, 96, 97, 105, 110, 112, 116 Cognitive load theory (CLT), 57, 58 Cognitive modeling, 32, 33 See also ACT-R, Keystroke-Level Model, GOMS Cognitive processes, 66 Cognitive walkthrough (CWT), 11, 83, 88, 95, 99, 104, 105, 109, 110, 112 Cogulator, 10 Color blindness, 39, 43, 45, 116 perception of, 43 vision, 38, 39, 43–45 Command Center, see Operation center Complexity, 4, 5, 23, 25–29, 31, 53, 58, 59, 99, 116 Comprehend, 4, 30, 40, 111 Comprehension, 25, 29–31, 40, 46, 53, 89, 90, 92, 113, 116 Control center, see Operation center Control room, see Operation center Critical systems, 1, 2, 6 © The Author(s) 2021 123 J. D. Oury, F. E. Ritter, Building Better Interfaces for Remote Autonomous Systems, Human–Computer Interaction Series, https://doi.org/10.1007/978-3-030-47775-2 Subject Index #### D Dashboard car, 23, 28 interfaces, 23, 28 Data entry date/time, 104 numeric data, 89 Decision making, 8, 25 Declarative knowledge, 32 Declarative memory, 32 Design choices, 6–10, 17, 18, 53, 57, 59, 89, 116 gold standard in design, 10 tradeoffs, 17–18, 24, 52, 60 guidelines, 2, 5, 22, 25, 30, 47, 50, 60, 87, 113, 115 principles, 2, 6, 16, 25–30, 38, 43, 46, 51, 52, 60, 64, 116 Designers, 2, 4–6, 8–10, 14, 16–19, 22, 23, 27, 28, 30, 31, 33, 37–44, 46, 47, 49–52, 55–57, 59, 60, 63–68, 87, 97, 104, 115 Disasters, 4, 26, 46 Distractions, 26, 28, 49, 50, 104 Divided attention, 49 See also Multitasking Driving, 23–25, 32, 49, 57, 58, 76 Dual-coding, 39 Dual-task, 33 See also Multi-tasking #### E Education, 19, 65, 67, 115 Emergent phenomenon, 37 Errors frequency, 26 implications for design, 31, 33 Event logs, 15, 74, 76, 81, 85, 86, 92, 96, 101, 103, 110, 111 Expertise, 31, 57, 67, 81 Expert users, 91 #### F False alarms, 18, 46 Fatigue, 26, 46, 59, 66 See also Attentional vigilance, Sleep Fatigue Avoidance Scheduling Tool (FAST), 19, 49, 52, 66 Feeling of knowing (FOK), 88, 89, 95, 112 Fixations, 40, 48 Flight, 46, 54, 55 Focus, 8–10, 13, 21, 24, 26, 31, 32, 38, 48–50, 83, 93, 96, 100 See also Attention Fonts, 5, 17, 24, 29, 39, 46, 92, 93, 116 Foundations for designing user-centered systems (FDUCS), 8, 88–92, 96, 100, 105, 106 Friendly fre, 24 Fundamental attribution error of design, 10 Funders, 7, 63, 78, 82 See also Stakeholders #### G Gauge (interface feature), 23, 28, 45, 89 See also Level indicator Gestalt theory, 41–43 Goals, 4–6, 11, 12, 22–25, 32, 33, 48, 53, 64, 71, 82, 88 Goals, operators, methods and selection rules (GOMS), 88, 91, 92, 94, 103–106, 108, 109 Gradient buttons, 107, 113 Guidelines, see Design guidelines #### H Habituate, 55 Help buttons, 90, 100, 107, 108 See also Tooltips Hick-Hyman law, 18 Hicks law, 18 High-stakes tasks, 50 See also Critical systems Human-computer interaction (HCI), 2, 4, 87, 89 Human error, see Error Human-system interface, 25 #### I Icons, 27–29, 46, 92, 95, 96, 105–108, 110, 113 Indicators, 23, 24, 39, 97, 113, 114 Interfaces, 2, 5, 7–9, 11–13, 16–19, 21–30, 33, 38–40, 43–47, 49, 51, 53, 54, 59, 60, 66–68, 71, 74, 79–83, 85, 87–89, 93–99, 103, 105, 109, 110, 112, 115 Interruptions, 30, 49–51, 55, 66, 95, 98, 116 See also Task-switching, multi-tasking, distractions, divided attention #### J Judgements, see Decision making Just noticeable difference (JND), 51 #### K Key, see Keystroke Keyboard shortcuts, 94, 99, 101, 104, 105 Keystroke, 12, 94, 104 Keystroke accelerators (KSAs), 91, 94 Keystroke-level model (KLM), 12, 112 See also Cognitive model Knowledge declarative, 32 procedural, 33 #### L #### M Managers, 2, 16, 63–65, 77, 78 Mars Water Detection System (WDS), see Water Detection System (WDS) Memory long-term, 32, 33, 55, 56 recall, 17, 91 recognition, 17, 91 short term, 92 working, 48 working memory capacity, 48, 56, 57, 59 working memory failure, 4 working memory load, 48, 57 Mental model failure, 4 Mental models, 4, 25, 28, 29, 31, 33, 37, 40, 43, 50, 54–56, 66, 68, 91, 101, 109 Mental workload See also Cognitive Load Menus, 18, 90, 91, 94, 104–107 Missing information, 27, 46, 116 Mission control center, 2 Models, 2, 8, 11, 17, 25, 31–33, 63, 98, 109 Multiple designs, see Prototyping Multi-tasking, 57 See also Interruptions, distractions, task-switching #### N NASA NASA Program Offce Scientist, 78, 80, 82–84 National Research Council, 8 Normalcy bias, 46 Note-taking, 30 Notifcations, 16, 77, 80, 84, 95–97 Novice users, 12, 90 #### O Operation Center (Op center), 2, 4–6, 8–10, 12, 13, 15, 16, 18, 25, 26, 33, 57, 63, 65, 78–82, 85, 87, 90, 95 Operators visual illusions, 38 OZ, 54, 55 #### P Paper, 9, 30, 67 Perception, 26–29, 31, 32, 38–40, 42–48, 55, 60, 66, 67, 91, 95, 97, 105, 111, 116 Phonological loop, 33, 57 See also Working memory Pilots, 26, 46, 53, 54 Popovers, 102–103 Pre-attentive visual processing, 40 Primary tasks, 23, 49–51, 53, 55, 57 Progress indicators, 89, 114, 115 Projection, 4, 31 Prototype, 13 Push buttons, 107, 108 #### R Radio buttons, 91, 107–109 Reaction times, 18, 19, 49, 66 Reading, 6, 17, 24, 39, 45, 46, 49, 55, 65, 67, 68, 91, 92 Recall, 17, 88 Redundancy, 15, 58, 75 Reliability, 4, 18, 67, 79 Remote autonomous systems, ix, 1–19, 85–106 Risk-driven incremental commitment model (RD-ICM), 6, 7, 10 Risks, 2, 4–11, 13, 14, 16–18, 21, 27, 28, 30, 43, 45, 49, 50, 57, 63–65, 79–82, 93, 97, 116 Risk-driven approach, see Risk-driven incremental commitment model Risk-driven spiral model, see Risk-driven incremental commitment model #### S Saccades, 40, 48 Safety critical systems, see Critical systems Screen, see Interface Scroll view, 103 Search felds, 102, 110, 111 Secondary tasks, 23, 49, 55, 57 Sensors, 9, 22, 38, 46, 73, 75 Shapes, 7, 25, 42 Sight, see Vision Signal detection theory, 18 habituation, 55 thresholds, 28 Situation awareness (SA) Stage 1: Perception, 26–29 Stage 2: Comprehension, 29–30 Stage 3: Projection, 31 Sleep, 49 Software engineer, 67, 80 Sound, 28, 51 Speed-accuracy tradeoff, 52 Speedometer, 23, 28 Spiral model, see Risk-Driven Incremental Commitment Model Splash screens, 90 Split views, 103 Stakeholder analysis, 78–82 Stakeholders, 2, 6–11, 17, 18, 25, 58, 63–65, 78, 80, 82, 83, 115 Stimulus, 25, 27, 29, 33, 38, 39, 41, 47, 48, 51, 116 Stimulus detection, 39 Stroop task, 38 Stress, 19, 50, 57, 59, 66 Studies, 2, 10, 11, 17, 18, 50, 52, 67, 83, 87, 93, 98, 109, 115 Sub-goals, 12 Subtasks, 11, 12, 81, 82 Supervisors, 14, 49, 78, 80, 86, 95 System failures, 2, 4, 10–12, 21, 27, 28, 43, 65, 82 System of systems, 1, 67 Systems developers, 6, 78, 80, 81 Systems engineers, 4, 16, 18, 40, 50, 58, 115 #### T Tab views, 104, 113 Tachometer, 23, 28 Tactile, 38 Task analysis (TA), 10–12, 46, 47, 71, 83, 88, 90 See also Keystroke-Level Model, cognitive walkthrough, GOMS Tasks, 2, 4–6, 8, 10–19, 22, 23, 25–27, 29–33, 37, 38, 40, 43, 46–50, 52–60, 63–68, 77, 78, 80–91, 94–97, 101, 103, 105, 108, 110, 112–116 Task-switching, 30, 48, 55 Tomahawk launch system, 64 Tooltips, 88, 92, 93 Training, 16, 17, 29, 50, 64, 77, 80, 90, 115 Types of memory, 10 Typing, 52, 111 #### U Usability, 2, 3, 8, 10–12, 63–65, 83, 88, 93 US Airways Flight 1549, 64 User-centered design (UCD), 2, 4, 6, 9, 17, 22–26, 87, 88 User-experience design (UX), 2, 4, 6, 8, 22, 102, 115 User feedback, 91 Users, 2, 4–8, 10–12, 16–19, 22–24, 27, 39, 40, 43, 45, 46, 48, 50–52, 54, 56–58, 63, 65–67, 71, 74, 78, 80, 82, 87–116 User-testing, 6 See also Study, A/B experiment USS Vincennes incident, 64 #### V Vigilance, see Attentional vigilance Vision pre-attentive visual processing, 40 visual processing, 40 visual search, 37, 39–40, 48 Visual illusions, 38 Visual processing, 37–41, 43, 50 Visual search, 40, 48 Visuospatial sketch pad, 33, 57 See also Cognitive load, mental workload #### W Wall of Screens, 16, 78, 79 Warnings, 15, 28, 30, 50, 77 Water detection system (WDS), 2, 12–16, 18, 19, 27–30, 46, 63, 71–87, 94, 97, 101 Working memory, 29, 33, 48, 50, 53, 55–60, 90, 91, 96, 103, 106, 109, 111 Workload, see Cognitive load, mental workload	doab	2025-04-07T03:56:59.014202	17-2-2021 09:54	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "006d7857-e96f-4630-aa40-deadb120d9aa", "url": "https://library.oapen.org/bitstream/20.500.12657/46794/1/2021_Book_BuildingBetterInterfacesForRem.pdf", "author": "Oury, Jacob D.\|\|Ritter, Frank E.", "title": "Building Better Interfaces for Remote Autonomous Systems", "publisher": "Springer Nature", "isbn": "", "section_idx": 0 }
006db864-879f-4bae-9f57-b9605ac62633	# The Gender Regime of Anti-Liberal Hungary Eva Fodor # The Gender Regime of Anti-Liberal Hungary "This authoritative analysis of anti-liberal Hungary's twenty-frst century twist on 'Kinder, Küche, Kirche' exposes its exploitation of women not only as unpaid carers in the home but also as a source of cheap paid labor. Fodor deftly links the Orbán regime's notorious opposition to gender equality to its embrace of pronatalism, xenophobia, and expanded funding for church-based childcare and eldercare. A terrifying, essential read." > —Ruth Milkman, City University of New York Graduate Center and author of On Gender, Labor, and Inequality (Illinois, 2016) and Immigrant Labor and the New Precariat (Polity 2020) "Eva Fodor's new book is an important contribution to the understanding of the worldwide drift from liberal democracy to anti-liberalism (or illiberalism) during the past 10–15 years. The study focuses on a case, a small country, which took a lead in this process: Hungary. A major contribution of the book is the description of the gender regime of anti-liberalism, what she calls a 'carefare regime.' While liberalism tends to commodify care work, anti-liberal regimes reemphasize women's role as mothers and housekeepers, and the poorest of the poor are turned into a 'female underclass' who have to keep their (usually miserable) market incomes and also deliver unpaid care services. Great book about anti-liberalism and especially its gender regime." > —Ivan Szelenyi, William Graham Sumner Emeritus Professor of Sociology, Yale University, USA "Finally, a book that offers brilliant insight into the seemingly perplexing: the gender politics of the contemporary Hungarian state. With sharpness and wit, Fodor reveals how the public attack on gender—indeed, even on the concept of gender itself—goes hand-in-hand with the rise of authoritarian rule and right-wing populism. Giving this new gender regime a name, 'the carefare state,' Fodor uncovers how it became so foundational to anti-liberal currents in Hungary. Part history of the present and part social policy analysis, The Gender Regime of Anti-Liberal Hungary makes an enormous contribution to understandings of anti-democratic politics, gender relations, and social inequality in Hungary and beyond." —Lynne Haney, Professor of Sociology, New York University, USA "Eva Fodor's The Gender Regime of Anti-Liberal Hungary insightfully dissects the anti-gender ideology of Prime Minister Viktor Orbán's government, which has eroded measures in support of gender equality and against gender-based violence, as well as the academic study of gender and sexuality. Fodor provocatively discusses a successor to neoliberalism's workfare, namely a 'carefare' regime in which child protection and women's paid and unpaid labor are regulated in the interest of preserving tradition and the Hungarian nation. Illustrating the gendered dynamics of carefare through a case study of fostering, Fodor's analysis is as somber and troubling as it is cautionary, telling a tale to which scholars and policy makers should carefully attend." > —Gail Kligman, Distinguished Professor of Sociology, University of California, Los Angeles, USA Eva Fodor # The Gender Regime of Anti-Liberal Hungary Eva Fodor Department of Gender Studies CEU Democracy Institute Central European University Budapest, Hungary #### ISBN 978-3-030-85311-2 ISBN 978-3-030-85312-9 (eBook) https://doi.org/10.1007/978-3-030-85312-9 © The Editor(s) (if applicable) and The Author(s) 2022. This book is an open access publication. Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland # Acknowledgments This book was researched and written amid a great deal of chaos and a series of crises, most of them political, some pandemic—related. Typically, this setup does not bode well for in-depth scholarly thinking but I am convinced that this case is an exception. I started this project in 2015 and did most of the data collection in 2015–2016. Then in the Fall of 2016, I was appointed Pro-Rector for Social Sciences and Humanities at the Central European University (CEU) in Budapest where I had been working for a decade. What seemed like a fairly peaceful administrative job in a small college changed overnight when in the Spring of 2017 the Hungarian government passed a modifcation of the higher education law, which made the operation of CEU in Budapest impossible. This was a straightforward political attack, part of the vilifcation campaign of the university's founder, George Soros. Given my high-level administrative position, I was thrust into the midst of the political struggle to save the university and keep it working in the place where it had deep roots and had been operating for 25 years, in Budapest. This put a quick stop to the progress of my research but gave me a frst-hand ethnographic view of sorts into the workings of anti-liberal power, especially as it targeted institutional academic freedom. Eventually, CEU lost the battle with the government and moved its graduate education to Vienna. While a ruling of the European Court of Justice in 2020 invalidated Hungary's modifed higher education law, the decision came two years too late to help. This would have been a different book had I not been able to witness and participate in this political struggle on a daily, hourly basis over the course of two years. In the process, my colleagues at CEU have taught me a great deal about politics, resistance, resilience and the sheer will to just go on and do the job of a university under all circumstances. I am eternally grateful. On a more technical but similarly grateful note, the open access publication of the book was made possible by the Provost's Open Access Publishing Fund at the Central European University. I owe heartfelt thanks to my over 80 respondents whose stories make up the argument in Chap. 3: foster parents, advisors, guardians and policy makers in the feld who allowed me access to their work through participant observation and interviews. They are doing admirable work and it was uplifting to be part of it even if for a short time only. Finally, I'd like to acknowledge my family—the three most important men in my life—without whom this book may never have been written because scholarly ideas, political struggle and life itself would have a very different meaning without them. # Contents # List of Figures # List of Tables # Orbánistan and the Anti-gender Rhetoric in Hungary Abstract This chapter introduces Hungary's anti-liberal political rule and its gender regime. It traces policy changes in Hungary since 2010, discusses the legacies of the state socialist gender regimes and the formation of a new, anti-liberal one. I introduce the term "carefare" and discuss how the concept of "gender" has been deployed by Hungarian politicians to legitimate an increase in women's unpaid care burden and their lack of attention to gender inequality in the labor market. I end the chapter with a description of my research methods and provide an outline for the rest of the book. Keywords Illiberalism • Anti-liberalism • Post-state socialism • Gender regime • Hungary • Anti-gender rhetoric Hungary is proof positive that history did not end, as Francis Fukuyama famously predicted, after the collapse of the state socialist regimes in the early 1990s. Within 20 years, the country became the poster child of democratic backsliding, right-wing populism and anti-liberal authoritarian rule, all combined with a capitalist economy whose operation oscillates between global neoliberal and eastward-looking neo-patrimonial principles. This novel form of governance is closely intertwined with a novel type of state gender regime—a combination of old and new elements in an exorbitantly patriarchal mix—which I call "carefare". This book describes the concept of carefare and its real-life manifestation in anti-liberal Hungary. Admittedly my argument is about a single country, but since elements of anti-liberal governance are gaining ground every day, the lessons here should serve as potential for comparison elsewhere. "There is no such thing as gender!" claimed a high-level Hungarian politician in response to critiques of the government's ban on masters' degrees in Gender Studies in 2018. And in denial, the term "gender" has been spread far and wide. After its landslide victory in 2010, one of the frst pieces of legislation Viktor Orbán's government introduced ended the requirement to eliminate gender stereotypes in the national curriculum of kindergartens. Soon the government moved from kindergarteners to a wide range of other social groups. The Parliament refused to ratify the "Istanbul Convention"1 by claiming offense at the word "gender" in the document, enshrined the requirement that families consist of a biological male and a biological female, passed numerous pieces of legislation which reformulated, restricted and rigidifed the social roles assigned to women and men, threatened women's reproductive rights, prohibited the option of sex change, as well as adoption by unmarried or non-heterosexual individuals. Members of the government have lobbied aggressively for the elimination of the term "gender" along with the concept of gender equality from policy proposals of international organizations. The government stopped funding NGOs which addressed gender equality problems and put an end to all government agencies that designed, implemented and monitored legislation to promote women. Through policies like these, accompanied by unabating government-inspired media propaganda, an anti-liberal gender regime has been constructed. Although this gender regime has its roots in the lengthy history of Hungary's varied past political orders, and carries some elements of both institutionalized gender relations in Western neoliberal democracies and the Central European state socialist gender regimes of the recent past, it is also distinctly different from both. Institutionalized patterns of gender relations or "gender regimes" are shaped by and themselves are constituent parts of political-economic governance (Connell 1987; Walby 2020). The literature on the <sup>1</sup>The "Istanbul Convention" or more precisely the "Council of Europe Convention on Preventing and Combating Violence Against Women and Domestic Violence" came into force in 2014. It was signed but not ratifed by Hungary. transformation of gender relations in anti-liberal regimes, especially in Hungary and Poland, has primarily focused on the ways in which the term "gender" has been used to create political mobilization and legitimacy, build internal loyalty and gather votes (Grzebalska and Pető 2018; Korolczuk and Graff 2018; Kováts 2020; Krizsán and Roggeband 2018). In this book I want to highlight different aspects of the newly emerging state gender regime: the transformation of women into carefare workers and the gender regime's—potential or real—impact not only on gender but also on class inequalities. Since 2010 the Hungarian government instituted policies and policy practice which offer a novel response to the "crisis of care" problem (Fraser 2016). In anti-liberal Hungary, care work is not commodifed, instead it is sentimentalized in a specifcally gendered way. It is not outsourced to poorly paid immigrant laborers, or widely available for purchase on the market from for-proft providers. It is also not offered—in suffcient quantity or quality—at a more moderate cost in institutions of care maintained by the state or by community-based non-governmental organizations. Instead, anti-liberal Hungary has been aggressively promoting the intensifcation of women's domestic care load through its all-encompassing pronatalism which ties social citizenship rights to having children, yet offers highly selective state support for the long-term work of caring for small children, even less for other forms of care. Simultaneously, women's paid work is required to maintain the family's subsistence (and often their access to state subsidies for children) but labor market gender inequality is openly embraced. Most women end up combining an increased volume of unpaid care work with long hours of full-time paid work in an economy that is shamelessly slated against those with care responsibilities. Compliance with such an exacerbated and unequal work burden is elicited through the rejection of gender equality as a principle, the elimination of alternative life courses for women, and a sustained political discourse which sentimentalizes and naturalizes women's care responsibility. Highlighting the analogies with workfare and prisonfare regimes of neoliberal capitalist economies (Peck 2001; Wacquant 2010), I call Hungary's state gender regime variant "carefare". Carefare, I argue serves as one of the main political, ideological and economic backbones of Hungary's antiliberal regime. # Hungary Since 2010: Building an Anti-Liberal State Right-wing, antidemocratic, authoritarian-leaning parties and governments have been emerging onto the international political scene in record numbers all over the world, but Hungary's case is one of the starkest. Hungary is the only European country whose democracy has been downgraded by the independent watchdog Freedom House to the category "Transitional or hybrid regime" and the only country classifed as only "partly free" within the EU (Freedom House 2021). Political scientists cite extreme party polarization (Enyedi 2016) and emerging populist tendencies (Rupnik 2016) as key enabling factors leading to democratic backsliding. The instability of democratic institutions after 1990, a pre-war tendency toward right-wing conservatism, the weakness of a professional middle class and the lack of a lively civil society will have also contributed to the ease with which Orbán's anti-liberal discourse and political rule gained ground. The economic trauma brought about by the collapse of the state socialist economy, the devastation of the ensuing economic crisis, the quick disillusionment with the unequal rewards of global neoliberal capitalism, which became especially evident during the 2008 economic recession were also important causal factors (Krastev 2016; Scheiring 2015, 2020). In this context, populist and nationalist ideologies promoted by Viktor Orbán and his party, FIDESZ–KDNP2 found fertile ground. The alliance won a landslide election victory in 2010 and started the work of building a new form of governance. Borrowing Fahreed Zakaria's term (Zakaria 1997), Orbán himself named his rule an "illiberal democracy" in 2014 and later a "Christian Democracy" in 2018. There is no consensus among political scientists about what aspect of this new form of governance is the most important or the most enduring. Some point to the dominance of a single party (Scheppele 2014), others emphasize its populist features (Enyedi 2016; Müller 2016). Bálint Magyar (2016) describes the regime as a "mafa state" where systemic corruption serves the interests of the political rulers and their loyalists. Other terminologies, emphasizing yet other features, include "hybrid regime" (Bozóki and Hegedűs 2018), "authoritarian capitalism" (Scheiring 2015), 2The full name is FIDESZ—Magyar Polgári Szövetség (FIDESZ—Hungarian Civil Association) in alliance with KDNP (Christian Democratic People's Party). The term FIDESZ itself is an abbreviation of the original name of the party until 1995, Fiatal Demokraták Szövetsége (Alliance of Young Democrats). I will call the ruling alliance FIDESZ–KDNP from here on. "neoliberal authoritarianism" (Szalai 2016), "managed illiberal democratic capitalism" (Szelenyi and Csillag 2015) or "national authoritarianism" (Kelemen 2017), to cite just a few. My emphasis when writing about gender relations is on the regime's radical and explicitly stated rejection of everyday liberal values, of the principle of equal opportunities, of tolerance of difference from a preconceived norm—hence I call it an anti-liberal regime. While there may not be a consensus about how best to name the regime, there is little doubt about the fact that during its more than a decade (so far) in power, Orbán's government has profoundly reshaped the principles and practice of political rule in the country: it has centralized and cemented its political power through legislative changes, decreased media freedom, freedom of speech and important forms of academic freedom, it has spent vast amounts of taxpayer's money on hatemongering propaganda to serve its political goals and created a regiment of loyal cadres through corruption and cronyism. At the same time, the government has followed many of the prescriptions of neoliberally minded structural adjustment policies: keeping the state defcit low, enforcing state austerity in areas where it would have served the vulnerable, weakening the rights of labor in successful bids to court foreign investors. It has also wasted resources from the European Union's structural funds and from the economic boom following the 2008 crisis by spending on frivolous investments designed to boost national pride and strengthen Hungarian identity, not to mention the personal wealth of select loyal followers, while neglecting to channel resources into health care, education or social support. It may be worth reviewing these policies in some detail before we proceed to discuss the gender regime built to support and service them. Upon gaining power the FIDESZ–KDNP government rewrote electoral rules in a way that favored the ruling party (Law CCIII/2011). But the government went well beyond familiar acts of gerrymandering: the new electoral law passed in 2011 cemented its power by making the decision on district boundaries pass as a "cardinal law" which could only be changed by two-thirds of the votes in Parliament. The new legislation turned the elections from a two-level system to a single-round one, and gave voting rights to typically right-leaning ethnic Hungarians living outside the borders of Hungary. These were some of the main actions taken to favor the incumbent party and guarantee its long-term hold on power (Scheppele 2014). Next, the government produced a new Constitution and the FIDESZ– KDNP dominated Parliament hastily passed it without much consultation or debate. Since the party had a two-third majority they did not need the support of opposition parties, so the procedure could be sped up. This new Constitution started the work of dismantling liberal democracy. It centralized power in the hands of the ruling party, weakened the power of the Constitutional Court and the role of the judiciary. The government made an attempt at replacing judges whom they considered too independent (Bánkuti et al. 2012). In the following decade the European Union (EU) initiated numerous legal procedures against Hungary to enforce the principles of the rule of law, none of which really steered the government off course. Orbán managed to resist even more radical attempts, such as when in 2020 the EU sought to include a clause in its budget that would require that countries abide by the principle of the rule of law in exchange for receiving EU funding. Hungary and Poland vetoed the budget proposal and the pandemic rescue package attached to it, which forced the Union to postpone and soften the measures and allowed Orbán to claim, yet again, "victory over Brussels". Numerous other formerly independent institutions, large and small, in areas from fnance to culture were gradually drawn under government control. To start, the government revised the media law and the vast majority of mainstream media were sold to government-friendly investors, who promptly steered them into a direction of absolute loyalty to the government. FIDESZ cadres were appointed as editors-in-chief, and journalists were expected to produce stories and accounts which corresponded closely to the message the government wanted to popularize. By 2018, several local media entities were united in a single conglomerate—a progovernment media empire of vast proportions. All national public television and radio channels, practically all regional papers and many internet outlets have essentially become propaganda machines reminiscent of the worst of the state socialist era (ATLO 2020). Cultural and academic freedoms have similarly been curbed. The government extended its fnancial and educational control over all state universities—in some cases more radically than in others. The self-governance of academic institutions was eliminated and the decision-making authority of the universities' Senate was replaced by the rule of governmentappointed committees. Other scholarly institutions suffered similar or worse fates: the rights and budgets of the Hungarian Academy of Sciences were signifcantly reduced and its research institutes were reorganized in a way that allowed political actors to have a vast infuence over the distribution of research funding. The government passed an amendment to the higher education law in 2017 specifcally targeting Central European University (CEU)—a US accredited graduate school founded, among others, by George Soros—and proceeded to force it to leave the country. Appeals to the European Union eventually led to a ruling of the European Court of Justice in favor of CEU, but the three years it took the Court to come to this conclusion made the decision moot. More granular-level interference into academic life has been a daily occurrence and incidents are too numerous to list here. One may be mentioned as it is of particular signifcance for the theme of this book: in 2018, the government unilaterally and without consultation or notice de-accredited gender studies MA degrees in the country. In addition to the media and educational/research institutions, nongovernmental organizations also came to be targeted by government ire: those which worked in areas that did not please the government were simply de-funded. Externally funded international NGOs have been suffering ongoing persecution. To retain a semblance of grassroots action, the government instead initiated and funds generously a loyal circle of "civil" organizations serving the party's agenda, and has been channeling vast amounts of money to a limited number of handpicked, loyal churches. Government-appointed loyal cadres manage these institutions in felds as diverse as the economy and fnancial oversight, through the judiciary, to the cultural feld, including managers of theaters, the national library and various museums, too numerous to count. In a fashion painfully reminiscent of the early days of the state socialist era, a new intellectual-professional upper class is in formation, and appointments depend primarily on loyalty rather than actual professional expertise or excellence in the given feld. These new cadres then receive a signifcant income through salaries and kickbacks from government funded projects as well as all the power of their offce, as long as they are willing to deliver what the government expects. Parallel to this is the creation via corruption of a wealthy upper class, a new bourgeoisie, whose economic prosperity depends solely on the amount of money they siphon off government- and EU-funded investments. Some of these assets remain in their own bank accounts, but most fnd their way back to the coffers of the ruling party. Corruption is extremely widespread and has increased precipitously in recent years in Hungary. This has been noted by practically all international agencies dealing with the issue (see, e.g., a report by GRECO 2020). The process of informal, illegal channeling of monies to individuals and politically loyal companies is built into the very core of the economic system, from the expectation that physicians get "tipped" in state hospitals to the Hungarian franchise of large software companies which receive kickback from state clients. Even proven obvious cases of corruption go unpursued and unpunished as Hungary's Attorney General, a loyal party cadre, is extremely reluctant to prosecute them. Notwithstanding its political populism and anti-liberal tendencies, the Orbán government did not altogether abandon all principles of neoliberal capitalism. FIDESZ–KDNP came to power in 2010 in the midst of the economic crisis and the economic performance of the country was weak in the frst few years. By 2013–2014, however, production picked up, as the international economic and fnancial context improved and European Union structural funds continued to pour in. By the middle of the second decade of the twenty-frst century, Hungary's economic growth outperformed EU averages, employment growth was striking, and real wages increased spectacularly in every year. Much of this success was based on Hungary's ability to attract foreign investors (Bandelj 2007) and these investments increased radically after EU membership. Well-known economists have argued that Hungary and other Central and East European countries may be net losers of EU membership, because multinational proft extraction is higher than the gains obtained via EU structural fund payments (Piketty 2018). Other accounts enumerate the various benefts multinational production has brought to the country and which cannot easily be expressed in cash payments (Meszaros 2018). For our purposes it is enough to note that the government's desire to attract foreign capital is undeniable and courting foreign direct investment has required creating a labor market structure where wages are low and reasonably trained workers are docile. In 2021 Hungary had the second lowest minimum wage in the EU overtaking only Bulgaria in this regard, and recent media accounts claim that over 40% of people do in fact work for the minimum wage, at least as per their formal employment contract. Although average wage levels signifcantly increased in the late 2010s, they still remain one of the lowest within the EU (Eurostat 2021a). And in recent years, the government has passed several other regulations with the goal of directly pleasing foreign investors and weakening labor rights: compulsory school age was reduced to 16 to feed the unskilled labor requirements of companies, the amount of overtime work an employer can require was increased by 25% and a great deal of fexibility was guaranteed to employers to compensate for the period, unemployment benefts were cut to almost nothing, the strike laws were changed in a way to make it more diffcult for the already weak Hungarian trade unions to organize, work hours were lengthened and vacation time cut in some sectors (Scheiring and Szombati 2019). Even in times of economic prosperity when wage growth was fairly steep and the state budget fush with money, the government neglected to channel resources into the three key areas which typically would increase the wellbeing of the population in a forward looking manner: health care, public education and social support. These, not coincidentally, are the sectors where female employment is especially high. While tax cuts and income linked benefts guaranteed higher income for the more advantaged, researchers note an ongoing stark state austerity in cash support targeting those at the bottom of the social hierarchy. In terms of health care, the glaring shortage of doctors, hospital beds and nursing staff, partly due to the out-migration of trained professionals because of the extremely low salaries and punitive work conditions, became clearly exposed during the coronavirus pandemic. In a period when economic growth leveled at around 4–5% annually Hungary spent less and less of its GDP on its already underfunded health care (Eurostat 2021b). Public education has suffered the same fate: teachers' salaries are exceptionally low, work hours are increasing, educational segregation is offcially endorsed and expenditure on education is declining in real value. The consequences are obvious: Hungary is doing poorly in international comparisons in life expectancy and especially healthy life expectancy, as well as in the performance of children in literacy and others skills tests. Hungary meets EU standards in access to childcare for children over three years of age, but is well below the recommended level for younger children. With the support of European Union funding, a small number of new nurseries have been built in the past years and others have been reclassifed into this category to boost the numbers in international statistics. Yet the quality of childcare varies: childcare workers receive close to the minimum wage in a lot of nurseries, and access to care is extremely uneven geographically. Parents who live in more prosperous areas may fnd it easier to get a spot for their child, while parents in rural areas must travel far to fnd care. Nowhere is austerity more obvious than in the allocation of social benefts. Unemployment benefts have been drastically cut in length and generosity and the government has sought to replace payment with public works programs. Social spending to support people in need has been reduced to the bare minimum, especially cash support for the vulnerable (Vastagh 2017). The only policy area where state spending has been boosted is "family protection", specifcally encouragement of and incentives for married working couples to have more children (Rat and Szikra 2018). I will discuss these measures, their logic and consequences in the next chapter. # Gender Regimes of the Past Orbán's anti-liberal government may be unique in the intensity of its single-minded pronatalism but women's social citizenship was, arguably, conditioned on their maternity in earlier times as well. In her now classic history of the Hungarian welfare state, Lynne Haney argues that from the late 1960s to the mid-1980s Hungarian women could make successful claims on the state on the basis of motherhood and protested vehemently the shift in emphasis toward material need as the main basis of claims making in the 1990s (Haney 1997, 2002). This is just one, albeit central, feature of the state socialist gender regime which explains the seamlessness of the transition toward anti-liberalism. There are at least three further areas where I see notable continuities between Hungary's state socialist legacy and our modern-day gender arrangements: women's historically high participation in gender segregated paid work, their concomitant, unchanged responsibility for care work of both the paid and the unpaid kind, and a general disdain for feminism and independent civil organizations fghting for women's rights. First, a word on the concept of "gender regime" is in order. A gender regime is constituted by "patterns of gender arrangements"(Connell 1987), which describe how members of a society are classifed into groups designated as men and women, the distinct social roles and responsibilities assigned to each, their symbolic representations, along with the inequalities built into these structures. All our social institutions are built on assumptions about and practices of gender: from the production process to reproduction, sexuality, institutions of politics and power, as well as emotions and cultural expressions. While gender may be manifested somewhat differently in each of these institutional arrangements, institutional gender regimes are deeply connected and in Connell's terminology cohere into a societal level "gender order", or following Walby's terminology, a new form of patriarchy or "gender regime" (Walby 2020). State socialist countries developed new patterns of gender arrangements after World War II under the leadership and supervision of the Soviet Union. At the time when, after the devastation of the war, Western European countries reveled in an increase in their birth rates and celebrated the stay-at-home housewife, women of all social classes were required to join the paid labor market in Hungary and in the other Central and East European states ruled by newly instituted communist parties. Women's paid work was understood as essential for women's emancipation according to the Marxist–Leninist doctrine. Not coincidentally, women's contributions was also sorely needed in the intensive industrialization project Central and Eastern European countries embarked upon after the destruction caused by World War II. Women's potential had been a vastly underutilized resource (Csányi 2019; Zimmermann 2010). The main goal of early women's emancipation policies was to cajole, and occasionally to force women to take up paid work. After World War II, about a third of all women had been working for wages in Hungary (Gyáni 1987) but by the end of the state socialist era this percentage climbed to over 75%. Similar rates were recorded in Nordic countries but, unlike there, in Hungary women worked full time all through their adult lives (Fodor 2021). Although employment levels plummeted after the collapse of state socialism and the attraction of the image of the middle-class stayat-home housewife featured prominently in the imagination of many overworked Hungarians, material reality only really allowed the very few at the top of the social hierarchy to drop out of work voluntarily. Women's fulltime labor force participation in Hungary has varied but remained high overall for the past 70 years, indeed higher than in most EU countries when expressed in full-time equivalency rates (OECD 2019). Inequalities at work were, however, rampant all through the state socialist period. Although data from the period are notoriously unreliable, researchers have found higher levels of occupational segregation in state socialist countries than in comparable capitalist ones during this period (Rosenfeld and Trappe 2002). In Hungary too, two-thirds of workers in sectors such as clothing or food production were women, and these jobs paid less than work in other sectors of the economy, which resulted in a wage gap that was measured at around 30% during the late state socialist era (Zimmermann 2010). This may have been somewhat compensated by the vast array of benefts in kind that workers received directly from the company they worked for, including kindergarten places, medical services, vacation home rentals and so on. Perhaps more importantly, housing—rented at a nominal cost from the state for life—was at least partly allocated by employers, and families with children as well as single parents enjoyed some privileges (Scheiring 2020). In the case of a divorce, mothers were typically granted full custody over children and, as a consequence, they also usually retained the right to the apartment. With a high divorce rate this led to diffcult life circumstances and/or increased homelessness for men, as well as some fnancial advantage for women. Similarly on the positive side, women's education attainment increased noticeably under state socialism and this, along with their growing work experience, led to their more equal share in positions of mid-level authority. Compared to neighboring Austria, which had started with a roughly similar gender regime after World War II but followed an altogether different route afterwards, women's labor market advancement in state socialist Hungary was signifcant (Fodor 2003). The need for women's paid work has a long history in Hungary, as does the unequal division of household labor. Communist parties proclaimed their intention to socialize child and elderly care as well as domestic work. After World War II kindergartens were opened, children and adults were offered subsidized meals in school and factory canteens, laundry facilities were available at a low cost in larger cities and so on. None of these proved suffcient, however, to ease women's reproductive burden to any signifcant degree, especially not in the context of a general shortage of services and goods required for the maintenance of a household. Women, as time budget surveys and ethnographic accounts attest, worked long hours in their paid jobs followed by a lengthy second shift at home (Ghodsee 2005). Some of the burden of reproductive work was alleviated by shared households. The proportion of multigenerational living arrangements was always higher in Central and Eastern Europe than in Western Europe and remained so, partly because housing shortages had affected the whole state socialist period. But mobility from the countryside to the cities also increased, and mobile families could not draw on the contribution of older generations in care work. It is thus not surprising that the birth rate, high after World War II, started to drop precipitously soon afterwards, and by 1959 Hungary's total fertility rate dipped below what would have been required to keep population levels stable. Partly in response to these problems and partly because intensive industrialization slowed by the mid-1960s, the logic of the communist party's women's emancipation policy shifted and even more of the care burden was moved to the realm of the family and onto the shoulders of women. Lengthy, paid maternity leave opportunities were instituted in almost all countries in the region in the mid-1960s. In Hungary, new mothers (and mothers only) could withdraw from paid work for up to three years upon childbirth and were guaranteed their jobs back upon return. Kindergarten places for children over three years of age as well as other social services related to childrearing became increasingly available and a balance was struck: it remained primarily women's responsibility to take care of children without much support from individual men but with some, typically in-kind, contributions from the state. In exchange, women continued to be employed as fulltime workers. However, working for wages in the socialist economy proved signifcantly less demanding than in its capitalist counterpart: work hours were shorter, overtime less frequent and the expectation of work intensity varied. "We pretend to work, they [the state] pretend to pay us" was the popular joke of the time and women's account of their work day often included doing the shopping in local shops or in the facilities within their work enterprises. Indeed, given the vast labor shortage and the political guarantee of a paid job for everyone, except for smaller pockets of the population, among them, for example, the Roma minority in Hungary, people could fairly easily fnd a new job if they found that the conditions in a specifc factory or offce were incompatible with their domestic responsibilities. Nevertheless, women worked signifcantly longer hours than men overall. To illustrate, a time budget survey taken in 1986 shows that among married couples with two children women spent 63 minutes a day on childcare and 227 minutes on domestic work for a total of 290 minutes. Fathers of two, on the other hand, dedicated only 61 minutes to these two types of activities combined. This 229-minute (almost 3.5 hours!) gap is not compensated for by the fact that men spent on average two more hours on doing paid work than women. Overall married mothers of two children had signifcantly less leisure time than similar men and a very long—almost ten-hour-workday altogether (KSH 2012). State socialist policies, except for a few initial steps in the early 1950s, mostly ignored the unfairness of this domestic division of labor. Disdain for feminism, Western women's movements and for the conceptualization of women's equality in terms of human rights is shared by anti-liberal and state socialist political regimes. While the notion of "women's emancipation" was an acceptable formulation in Marxist–Leninist ideology, those working toward it could under no condition be called "feminists" (Barna et al. 2018). State socialist policy makers banned Western feminist literature, and confscated it when they found intellectuals trying to smuggle it into the country. Achieving gender equality may have been an oft-repeated political goal, but it had to be initiated and carried out on the terms of the Communist Party. Women's grassroots organizations, numerous before World War II, were replaced by the Hungarian National Women's Association, which historians argue, had some degree of independence but was far from a true representative of women's interests nationally, and certainly did not invite a diversity of women's voices to be heard (De Haan 2010; Funk 2014; Ghodsee and Mead 2018). It is not surprising, therefore, that when Hungary joined the European Union, gender mainstreaming measures enforced by the accession requirements were not widely welcome (Kováts 2020). Indeed, in international surveys Hungarians tended to express more conservative gender role attitudes than citizens of other countries in the Union (Pongracz 2005). Without much conviction, the socialist government of Hungary introduced the necessary regulations and set up the required institutions that monitored the main indicators of gender equality, but popular support for the term or for women's struggle in general was negligible. Small feminist groups worked toward specifc goals, including violence against women, or reproductive rights, but funding primarily came from international organizations as did, often, the specifc agenda and discourse (Fabian 2014). The EU's gender mainstreaming policies did not gain widespread popularity (Ghodsee 2005; Gregor and Grzebalska 2016). In sum, women's ongoing participation in paid work was necessary in both state socialist and post-state socialist gender regimes, but little real effort was made at redistributing or socializing care work. Not unrelated to this, neither the state socialist emancipation effort nor the EU's haphazardly enforced and rather limited gender mainstreaming agenda generated much enthusiasm for the concept of gender equality. It is no wonder that in 2010 Orbán's new government could simply dismiss with impunity both the rhetoric and the reality of gender equality policies and assemble a new form of gender regime to support anti-liberal state building. # An Anti-Liberal Gender Regime But is this anti-liberal gender regime really new? I have identifed a number of continuities with Hungary's state socialist past and many of the policies I describe in the next chapter will be familiar from there or from elsewhere in the world. Sylvia Walby, for one, argues that the gender regimes of authoritarian states are not necessarily unique. Walby (2020) traces the evolution of gender regimes (or forms of patriarchy) from what she calls "domestic" to "public", and distinguishes at least two varieties of modern public gender regimes: neoliberal and social-democratic. She acknowledges the authoritarian turn in European politics but claims that authoritarianism is easily compatible with neoliberalism, so gender regimes of authoritarian states do not necessarily constitute a unique form. In a similar vein, Mudde and Kaltwasser (2015) claim that populist governments have diverse gender ideologies, left-wing in Latin-America, rightwing in Western Europe, hence, they argue, the position of populists on gender issues is unclear and depends on the national context, it is not a unique variety of gender regimes. Perhaps what we are witnessing is simply a turn toward conservatism, characteristic of neo-patrimonial regimes, such as Orbán's or Putin's (Szelenyi and Csillag 2015)? Or the continuation of the state socialist legacy of gender inequality? Csányi (2019) connects the new regime's emphasis on traditional gender roles with the exploitation of women's cheap labor. He emphasizes the continuities from the 1950s, and describes the novelties emerging after 2010 as primarily in the realms of cultural representations. Or perhaps inconsistency and ambiguity are the defning features of Orbán's gender policies (Kováts 2020; Szikra 2018)? Yet others emphasize the coherence and uniqueness in the institutionalization of gender inequality in the social fabric of countries which deny their allegiance to liberal democracy. Historical accounts, for example, point to similarities with "conservative authoritarian gender regimes" in Japan and Germany of the past (Shire and Nemoto 2020). Grzebalska and Pető (2018) claim that Hungary's and Poland's authoritarian governments have a unique "modus operandi" closely tied to their gender ideologies, and highlight the foregrounding and mainstreaming of the family rather than gender equality policies (Juhász 2012), the appropriation of the space for fghting for gender equality, and the use of an anti-gender rhetoric to gather all political enemies under one umbrella (Kováts and Poim 2015). Krizsán and Roggeband (2018) also point to the closure of civil space for a gender equality agenda, the relationship between nativism and nationalism in policy making, and the weakening and elimination of women's movements as common features of gender regimes in illiberal states. I side with those who see a new variety of gender regime emerging. I argue that since the mid-2010s the government has redefned the problem of care into one of demographic decline and proceeded to pass a policy package using principles of "carefare". Carefare policies—to be described in more detail in the next chapter—discipline women into accepting an increased unpaid care work burden combined with unequal treatment in the labor market in exchange for economic survival or, in some cases, slight improvements in the fnancial position of their families. The government's gender policies aim to reorganize not just gender relations but social stratifcation itself by trickling down some limited resources to select "deserving" social groups, whose contributions to the economy is essential and whose votes and political loyalty the government is counting on. To legitimate these policies, the government increasingly relies on the rhetoric of global "anti-gender" movements. # Setting the Stage for State Mandated Patriarchy: Anti-Gender Discourse in Hungary Numerous authors describe the global spread of the rhetoric against "gender ideology" and its important role in offcial political communication in Poland and Hungary (Korolczuk and Graff 2018; Kováts 2018; Kováts and Pető2017; Kováts and Poim 2015). "Gender" in this context has come to signify political issues and affliations well beyond the actual reality of gender relations. In Hungary too, the Orbán government has successfully divorced the term "gender" from actual policies about women's and men's social participation and turned it into a frenzied political rallying cry. It is not alone in these efforts. Democratic backsliding in several countries (from Poland, Romania to Brazil and beyond) has been accompanied by state-sponsored propaganda which denies the usefulness of the concept "gender" in regulating women's and men's role in society (Kováts 2018; Kuhar and Paternotte 2017). This "anti-gender" discourse has common themes but a different focus in different countries, a phenomenon, which supports the argument that it is primarily a political tool, a "symbolic glue" which holds allies, enemies and topical political themes together (Kováts and Poim 2015). I will briefy indicate a few elements of the Hungarian variety here and argue that, even though the discourse is not about gender relations, the use of this rhetoric has a lot to do with gender: it sets the stage for ignoring gender equality policies and reinforcing state mandated gender inequality. Fig. 1.1 The number of articles which mention the term "gender" in Magyar Idők/Magyar Nemzet Hungary is a relative late comer to the anti-gender scene, but the deployment of the concept of "gender" has accelerated in the past few years and is fercely ongoing at the time of writing this book. Even a cursory analysis of a FIDESZ-owned, self-proclaimed pro-government national newspaper, Magyar Idok̋ (in English Hungarian Times, later renamed Magyar Nemzet, or Hungarian Nation) illustrates this point. As Fig. 1.1 shows, Magyar Idok̋'s online portal published only 20 articles which used the term "gender" in 2015, while by 2020 a whopping 281 appeared in the paper. (Another pro-government outlet, Origo, also increased its attention to gender going from publishing 18 articles, which mention the term "gender" in 2017, to 32 articles in 2018, to 50 in 2019 and 65 in 2020.) Magyar Idok̋/Magyar Nemzet's output represents a more than ten-fold increase within a fve-year period between 2015 and 2020 and one which meant that the term gender has been used almost daily on the portal for the past three years. The government whose self-proclaimed goal is to eliminate "gender", and whose representatives deny its very existence, nevertheless deploys the term more often than has any previous government before them. Fig. 1.2 Appearance of key terms in articles which contain the word "gender", 2018–2020 (in percentages) This increased usage does not mean, however, an intensifcation in public discussions about gender relations. Partly in an effort to avoid just that, the government has emptied the meaning of "gender" as a concept signifying relationships and systemic inequalities between men and women, and has repurposed it for use to distinguish and legitimize its political agenda. I read, coded and analyzed 156 articles published on the online portal of the newspaper in the months of February, May and December of 2018, 2019 and 2020 to get a sense of the government's message and overall understanding of the term "gender". During these three years the term gender almost never referred to relationships between men and women. Indeed, as Fig. 1.2 shows, individual women or even women as a group are practically never mentioned in the 155 articles, and in only 15, fewer than 10%, is there any discussion of gender inequality or women's social position. Most of these 15 address issues of violence, only 3 mention labor market status or social welfare. Even motherhood or parenting is not on the agenda, only the abstract term "family" features with regularity. Instead, "gender" was most prominently used during these three years to weave a story about migration and Hungary's struggle against the European Union's migration quota. Figure 1.2 demonstrates this claim. In 2018 43.6%, in 2019 41.4% and in 2020 47.1% of all articles which contained the term "gender" also mentioned migrations and migrants. For example, Magyar Nemzet expresses concern about what it sees as "the aggressive propaganda about gender and migration" (December 26, 2020) threatening the integrity of the Hungarian nation in one of its articles during the Christmas period. In a similar vein, an article two weeks later assures the public that "Hungary … resists the integration of masses of migrants and the gender craze" (Jan. 13, 2021) emanating from the West. The European Union features prominently: 44% of all articles which mention the term "gender" also cite the EU, typically as a pro-gender enemy of the Hungarian nation. A single example suffces to illustrate this from an article published on December 14, 2020: "I was reminded: the gender lobby is hard at work and as part of the migration action plan 34 million migrants will get voting rights in the EU" (December 14, 2020). The relationship between the "gender lobby" and the "migration lobby" is not made explicit, the two are used simultaneously, indicating that they are the same or at least the same people are behind both. The blame for the EU's migration policy falls on the shoulder of the "gender lobby". Along the lines of what researchers have described elsewhere gender policies or gender ideology is simply used as a way to identify the "enemy", the "other side" or "left-liberal forces", specifcally those who seek to impose migrants on the country (ibid.). Gender ideology—if defned—refers to the acceptance of transgender people as legitimate members of society. The Hungarian government and its propaganda machines are openly and increasingly homophobic and reject all forms of sexual identities which are not hetero. Homosexuality is in fact seen as analogous to the problem of migration: several of the EU's norms, including those related to gender and migration, are understood as detrimental to Hungarian's values and as externally imposed and alien (Korolczuk and Graff 2018). Increasingly during the three years, stories about sex changes and variability in gender identity are problematized, laying the groundwork for various policies on the theme, including Hungary's ban on sex change, the prohibition of adoption by nonheterosexuals or the reinforcement of gender stereotypes in education. It is no wonder that OECD statistics from 2018 show that Hungarians are the least tolerant of all OECD countries toward ethnic, sexual and religious minorities, a sharp increase from 2008 when the views expressed by Hungarians were no different from the average. Gender is one, although not the only, political tool to construct an external enemy and mobilize for resistance against it. The concept of "gender" is thus used by Hungary's top-level politicians and government-controlled media to reinforce, communicate and persuade the population about their anti-liberal agenda. Depicted as a "foreign" concept—hence the use of the English term gender—it is steadfastly associated with other themes in the government's political repertoire and is described as dangerous to Hungarians, indeed to the fate of civilization itself. The government is thus tasked to reject these agents of evil and, as David against the Goliath of Brussels, to fght for the ultimate good of all against the "gender lobbies", "genderism" and the "gender ideology". Indeed, much of this discourse is not directly about gender or gender equality. But it does serve the purpose of taking attention away from gender equality policies and the possibility of claiming rights for women as women. The government has successfully tied the concept of gender equality to "liberalism", that is, politicized it and associated it with a specifc side of the political spectrum. As it is rejecting liberalism, it can thus legitimately and without further explanation reject gender equality policies as well. Note the close association between "gender ideology" and "liberal open society" in the text of Hungary's Minister of Justice, Judit Varga in a Facebook post on February 1, 2021: "We experience with great concern the breakthrough of the liberal open society. … Religion, nation, traditional family model … traditions have no place in that. … Instead, there is gender ideology, Christian persecution, a technological dictatorship of opinion, destruction of nations, and the creation of a grey uniform society in which everyone must be liberal and an individuality" (the translation is from the original post available online). # Outline of the Book and a Brief Explanation of Research Methods This book utilizes original data from several research projects I have conducted over the past six years. Chapter 2 explains the concept of carefare: its manifestations in social policy and political discourse, as well as some of its consequences for social and especially gender inequalities. To make the case I primarily rely on data from aggregate sources, such as Hungary's Central Statistical Offce, OECD and Eurostat datasets. In addition, I present results from statistical analyses of data from the dataset EU SILC from the years 2011 and 2017. The European Union Statistics on Income and Living Conditions (EU SILC) is a harmonized, annually collected EU-wide survey with a large enough case number to allow the analysis of smaller social groups. I use it to measure what is called the "motherhood penalty" in Hungary and its change over time. The third form of data come from a series of interviews I conducted with my colleague, Christy Glass over a decade-long exploration of the motherhood penalty in Hungarian frms, the nuances and conditions of its application, as well as how it is experienced (Fodor et al. 2019; Fodor and Glass 2018; Glass and Fodor 2007, 2011, 2018). The most recent series of interviews took place in 2019 when we talked with 24 mothers working in state administration, who responded to questions about the cut in vacation days and the lengthening of work hours, measures introduced simultaneously with the government's new pronatalist family policies. We sought to fnd out how this group of young professional women, who are clearly targeted by the family policies, evaluate their signifcance from their own points of view. Finally, in Chap. 2 I also present data from my analysis of a document published on the occasion of the International Women's Day in 2019 by a government funded organization called FICSAK, "Organization for Young Families" (FICSAK 2019). In this heavily subsidized and promoted booklet, high-level Hungarian male politicians and a handful of public personalities offer greetings to women to celebrate the occasion. I only used the quotes from politicians, ignoring otherwise famed participants, but politicians represented the majority. Each quote—altogether 90 of them—is a few paragraphs long and they are collected in a booklet entitled Women's Soul as Seen Through Male Eyes. These texts highlight better than most other documents what role is assigned to women, what achievements and character traits are praised most by top-level policy makers. I coded the quotes by theme and will cite the relevant sections in the next chapter. Chapter 3 describes how principles of carefare are realized in practice in the special context of Hungary's child protection system, and specifcally among foster care workers. Orbán's government transformed this area of social service through a new piece of legislation in 2014 and the process and its outcome illustrate brilliantly several aspects of the workings of a carefare state. I started studying the work of foster parents in 2015. During the past six years I conducted over 80 interviews with various actors in the foster care system, most importantly with 52 foster parents living in Pest county, in and near Budapest, as well as in a handful of small towns and villages in the north-east of Hungary. These areas are two of the most populous in terms of foster families. They are also quite different: I interviewed in the poorest and ethnically most diverse region of Hungary as well as in better-off areas around the capital. I initially got in touch with foster parents through their agencies, so I frst talked to those who were preselected possibly for their performance or easy collaboration with the agency. But I gained further contacts from the foster parents themselves so I could broaden the circle of interviewees. Nevertheless, I most likely conducted interviews with foster parents who were generally satisfed with and proud of their work, in addition to a few who held a major grudge and wanted to talk about that. Most interviews were conducted in the home of the foster parent and lasted between 90 and 180 minutes. Foster parents are used to having strangers in their homes who ask about their lives, so they have also developed strategies to respond in a way that preserves their privacy and dignity. But most people who visit them ask about their children and I was interested in them as workers, their daily routines, their experience, their lives and choices. This was a novel experience for many who felt underappreciated as workers and parents, so they typically welcomed the chance to talk. I taped and transcribed the interviews and analyzed them using the software NVivo. Names and minor demographic details have been changed to preserve anonymity. In addition to talking to foster parents, I conducted regular participant observations in two large foster parent agencies during birth-parent-foster child visitations, and helped out at other events at one of the agencies during the years of 2015–2016. I interviewed over 30 foster care advisors and various actors in the child protection system, including policy experts, managers of foster parent networks and politicians in Ministries who were in charge of the transformation of the system. I obtained data from the Central Statistical Offce, the Hungarian Treasury and the Ministry of Economics. Experts and policy makers provided me with numerous documents which were also used for the analysis. Next, let's explore the concept of carefare. # References ATLO. 2020. A magyar média elmúlt tíz éve (The Past Ten Years of the Hungarian Media). Budapest: Atlatszo - CEU CMDS. https://drive.google.com/fle/ d/1JafkHaGiF1tlFwUiS1FQsSz1uuypD8D5/view. Bandelj, Nina. 2007. From Communists to Foreign Capitalists: The Social Foundation of Foreign Direct Investment in Postsocialist Europe. Princeton, NJ: Princeton University Press. Connell, R.W. 1987. Gender and Power. Cambridge, MA: Polity Press. ———. 2021. Gender Regimes. In Central and East European Politics: Changes and Challenges, ed. Zsuzsa Csergo, Daine Egletis, and Paula Pickering, 5th ed. Lanham, MD: Rowman and Littlefeld. Kováts, Eszter. 2018. Questioning Consensuses: Right-Wing Populism, Anti-Populism, and the Threat of 'Gender Ideology'. Sociological Research Online 23 (2): 528–538. ———. 2020. Post-Socialist Conditions and the Orbán Government's Gender Politics Between 2010 and 2019 in Hungary. In Right-Wing Populism and Gender, ed. Gabriele Dietze and Julia Roth, 75–100. Bielefeld: Transcript-Verlag. ———. 2020. The Retreat of Liberal Democracy: Authoritarian Capitalism and the Accumulative State in Hungary. Challenges to Democracy in the 21st Century. London: Palgrave Macmillan. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapterss Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # A Carefare Regime Abstract Hungary's anti-liberal government has invented a novel solution to the care crisis, which I call a "carefare regime". This chapter describes four key features of the policies, policy practice and discourse that make up Hungary's carefare regime. I argue that in contrast to welfare state models familiar from developed democracies, in post-2010 Hungary, women's claims to social citizenship are most successfully made on the basis of doing care work. The state is re-engineered rather retrenched: services are not commodifed but "churchifed" in an effort to redistribute resources and build political loyalty. Women are constructed as "naturally" responsible for reproduction and care and this responsibility is tied to sentimentalized notions about femininity and true womanhood. In addition to providing care in the household, women are increasingly engaged in the paid labor market too, where the tolerance for gender inequality is offcially mandated. A carefare regime provides limited fnancial advantages for a select group of women, while simultaneously increasing their devalued work burden both in and outside the household: it feeds a growing underclass of women workers. Keywords Care crisis • Welfare regimes • De-familialization • Re-familialization • Carefare • Wage gap • Labor market • Gender inequality • Social citizenship • Demography • Family policies "And I hope that you—yes, you—have the ambition to lean in to your career and run the world. Because the world needs you to change it." Sheryl Sandberg, Lean in: Women, Work and the Will to Lead (2013) "Do not believe that we, women must always compete with men. Do not believe that we must compete in every moment of our lives, or that we must have the same position, the same salary as them … let's be happy that we were born women. … Let's be happy that we were given the gift of being able to love and take care of others." Katalin Novák, Minister for Families in Hungary in a video message to women, (HVG 2020) Over the past decade and a half the problem of the care crisis has gained traction in both the academic literature and in popular media (Ehrenreich and Hochschield 2004; Rosen 2007; Williams 2018). Recently, Nancy Fraser (2016) has argued emphatically that a "crisis of care" was looming in western capitalist societies caused by the increasingly unresolvable confict between the logic of social reproduction and that of aggressive capital accumulation. Fraser points out that although capitalist production requires healthy, socially apt and highly skilled human beings to meet its ever-growing proft targets, the work that goes into producing these individuals and the communities in which they thrive, that is, the work of social reproduction, has become devalued to the extreme by the very same logic of accumulation. Financialized neoliberal capitalism, she claims, has aggravated the above contradiction to the point of an inevitable explosion, although Fraser muses about the possibility of the emergence of creative "mutant" regimes, which would offer temporary solutions to patch up evolving crisis tendencies. Hungary's anti-liberal gender regime is such a mutant. I argue that since the mid-2010s the Hungarian government has been offering a novel response to the care crisis, one which is successful in generating a suffcient degree of political legitimacy even in the face of growing inequalities and social disintegration. I call Hungary's anti-liberal solution a "carefare" regime. Carefare is a form of state response to the care crisis, a set of social policies, policy implementation and related discourse within an anti-liberal political culture and an authoritarian capitalist economy. Carefare is not the only possible anti-liberal response to the crisis of care, but it is certainly one of them and a successful mutant at least in the short run. Below I discuss in detail four features which jointly distinguish "carefare", Hungary's response to the crisis of care, from the generic model of Table 2.1 A comparison of the logics of carefare versus the "two-earner family" model the "two-earner family", found, with variations, in numerous western countries guided by principles of neoliberalization (Fraser 2016; Walby 2020). None of the elements of carefare are particularly new. Indeed, several authors point out the direct links and similarities between elements of anti-liberal and neoliberal governance (Jessop 2019; Scheiring 2020). Yet the combination of these features results in a unique discursive interpretation of the care problem and a set of social policies, which do, in fact represent a novel answer. Table 2.1 below summarizes the four set of features in comparison. After a short discussion of the history of anxieties about the size of the population in Hungary, I explain and provide evidence for each in the rest of this chapter. First, in a neoliberal capitalist welfare state, successful claims on the state are made on the basis of social insurance and, secondarily, material need. These constitute the bases of social citizenship. Doing unpaid care work does not carry a social insurance scheme, and claims to the state cannot be made on the basis of raising children, or being good parents. This is indeed the crux of the confict between production and reproduction (Fraser 2016). In Hungary, however, care work has become a centrally important axis of social citizenship claims. Second, anti-liberal states are powerful and extensive, unlike their neoliberal counterparts which tend to disinvest in general and in social reproduction in particular. Hungary's anti-liberal state has increased already generous funding for several important aspects of social reproduction. In addition, instead of commodifying and marketizing previously state provided services in an effort to cut costs, it directs centralized state funding in a way as to maximize not proft or cost-cutting but political loyalty. Third, in Hungary care for children and the elderly is enthusiastically and selectively familialized. State policies assign care work to women and identify the heterosexual family home as its principal location. In neoliberal regimes, care work is increasingly outsourced to vulnerable, often immigrant domestic workers, to marketbased providers, and/or to state institutions. The main focus of policy objectives is typically to ensure that native, working age women do less care work, while in Hungary the opposite is the case. A political discourse which associates care with women's "natural" essence supports this process. Fourth, in addition to doing care work in the home, women are also incentivized to do waged work. This is done through tying reproduction related social benefts to work history and family income levels in addition to care work. This necessitates a dual earner couple. But state provisions do not address work–life balance problems, and gender equality measures are summarily rejected by Hungary's anti-liberal political leaders. A truly vicious trade-off is emerging where women must accept inferior work conditions in exchange for the possibility to meet care responsibilities. This is especially problematic for women in the lower educational brackets and those living in rural areas where work options are limited. One consequence of carefare is obvious: it increases women's work burden while leaving men's untouched. In addition, carefare reinforces and exacerbates class inequalities because better-off families can utilize more of the income-based provisions than those with lower wages. At the same time, however, the logic of carefare rearranges patterns of socio-economic disadvantage at the bottom of the social hierarchy too. Pronatalist provisions boost the wellbeing of specifc working class groups: those who have several children and some semblance of formal employment. These families have suffered vast social disadvantages in the past. Carefare promotes them into the category of "deserving families" and legitimizes their successful claims to social benefts. It is to these families that some of the economic growth gains and EU funding trickle down, even amidst growing overall social inequalities. If authoritarian policies serve as a political strategy to overcome conficts generated by the growing inequalities of neoliberal capitalism (Scheiring 2020), carefare serves the same purpose via different means. # "Care Crisis" as a Demographic Crisis in Hungary Hungarian policy makers have long acknowledged a pervasive crisis of care but only in one area of reproduction: the declining number of births to Hungarian women which they closely associated with the impending death of the Hungarian nation. Indeed, defning the declining birth rate of the native population as a "demographic crisis" is well known in European Union-wide thinking as well, although policy recommendations from the EU include a variety of measures which could potentially reduce the burden of care work on women. As I will show below this was not the direction the Hungarian government took. To understand this rather narrow re-conceptualization of the problem of care, we need to understand the lengthy history of deep-seated anxieties about the size of the population in the country. Hungary is a small country of about 10 million people and this number has been shrinking steadily since 1981. The total fertility rate (TFR) has not reached what is typically considered suffcient for reproduction since 1959 (KSH 2019a). In this regard, Hungary's demographic characteristics are quite similar to those of many other post-socialist countries, which started to experience a decline in births in the 1950s. This then continued steadily throughout the twentieth century with a sharper drop in the fertility rate in the early 1990s after the collapse of state socialism, which brought about a deep economic crisis and major societal upheaval. Two decades later, the recession of 2008 produced even more societal stress all over the region and resulted in a further decline in fertility in the frst decade of the twenty-frst century. Soon, however, total fertility rates began to increase in all poststate socialist countries, with Hungary lagging somewhat behind, but picking up speed by 2013. In 2019, Hungary's total fertility rate was in line with that of the EU 27, even if the number of births had not shown a similar increase due to the smaller size of the cohort in reproductive age in the late 2010s (Eurostat 2018a; KSH 2019a). Although Hungary's fertility rate is not signifcantly different from that of other Central and East European countries, or even the EU 27 average, the country's crude death rate is one of the highest in the European Union, and life expectancy, as well as healthy life expectancy, is one of the lowest (Eurostat 2018b). Indeed, all Visegrád 4 countries exceed Hungary in life expectancy and only Romanian and Bulgarian women die younger than Hungarians within the Union (ibid.). On the plus side, this is one of the reasons why Hungary's old age dependency ratio is somewhat lower than the EU average of roughly 30%. Hungarian policy makers have mostly ignored the problem of early deaths and conceptualized the demographic crisis primarily as a threat to Hungarian nationhood and only secondarily as a potential human resource shortage. # Anxieties About Populations Biopolitical concerns about the size and quality of the population have long plagued Hungarian political discourse (Melegh 2019). The country lost a great deal of its territories and over half of its population following World War I—some to the damages of the war itself, most to the dictates of the post-war Treaty of Trianon. The pain of the war loss intensifed debates about demography and specifcally about the phenomenon of the "single child" popular in rural households in certain regions in Hungary (Andorka 1975). The anxiety about the size of the population was explicitly connected by politicians, writers and public fgures alike with the future of the Hungarian nation and the looming threat of German invasion (Heller et al. 2015). After World War II even though war losses were signifcant, discussions about demography were silenced for a while, as issues related to industrialization, war recovery and an ideological commitment to the fght of the international rather than the national proletariat were considered more important by the leaders of the Communist Party. However, by the middle of the 1950s, when the post-war baby boom failed to materialize in Hungary, the government decided to take a radical step and banned abortions altogether (Pongráczné 1999). The number of births increased temporarily but political pressure forced the government to abandon the measure in 1956. In the absence of other types of contraceptives, abortion became the primary form of birth control for Hungarian women: between 1960 and 1973 the number of abortions, legal and accessible, exceeded the number of births (KSH 2019a). After the revolution of 1956, the Hungarian government's population policy started to lean toward incentives rather than prohibition. In 1967, a three-year paid maternity leave was introduced as a way to encourage births and also to regulate the labor market. But as a backlash against permissive abortion regulations and the extremely high number of procedures, the government created a new population policy in the early 1970s, which added a set of restrictive measures as well. Around this time an extensive public debate took place on the pages of Hungarian magazines and weeklies. Public fgures, writers, sociologists and demographers expressed concern about the developments in population trend, linking the problem of fertility decline to classic tropes ranging from the death of the nation and the disappearance of Hungarians from the planet, to moral concerns about abortions, the idyllic image of large healthy families of the past, as well as to the relationship between women's emancipation and labor force participation and their willingness, inclination and ability to produce more children (Heller et al. 2015). Current defnitions of the problem of care resonate deeply with many of these ideas. Population concerns were not limited to Hungary, of course, but in the 1970s and 1980s the primary global concern was the "population bomb" rather than depopulation. It was after the turn of the twenty-frst century that the issue of demography came to the attention of policy makers in the European Union.1 As the frst cohort of baby boomers entered retirement age and looked forward to decades of happy retirement, the notion of old age dependency came to be conceptualized as a looming problem. The size of the EU's population started to shrink in 2015 prompting further discussions about depopulation, the cost of aging and population projections. Although nationhood, national identity and demography had been points of interest for the Orbán government immediately after its accession to power in 2010, it was the refugee crisis of 2015 which cast the population problem in an altogether different light and allowed it to gain the political momentum to profoundly change social policy. In the summer of 2015, a large wave of refugees entered Hungary. In average years about 2000–3000 people sought asylum in the country, by early fall of 2015 the number was close to 180,000 (KSH 2019b). Refugees from Afghanistan, Syria, Pakistan, Iraq and Kosovo arrived in Budapest, submitted their request for asylum status but moved on toward more prosperous and inclusive parts of Europe, such as Germany, France or the UK. The food of asylum seekers entering the EU through the Serbian–Hungarian border took the country by surprise and no humanitarian support was <sup>1</sup> I want to thank Zsolt Spéder (head of the Hungarian Demographic Research Institute) for his lengthy consultation with me on topics related to population policy in Hungary and globally. See also Melegh (2019). forthcoming. Instead, the Hungarian government constructed the notion of the "refugee crisis", built a wall to restrict entry on the southern border and started a country-wide propaganda campaign which depicted refugees and migrants as potential terrorists and threats to the social and cultural wellbeing of all. A second propaganda campaign demonized George Soros, and argued that he, with the complicity—even direct assistance—of key politicians in the European Union encouraged and funded migration in an effort to destroy the purity of European Christianity. Hungarian politicians were not ashamed of their openly racist and xenophobic messaging which had the expected infuence: Hungarians developed a real and measurable fear of the person of the "refugee", even though most of them had never actually met one in their lives. Within three years, the number of asylum seekers plummeted to levels well below those pre-2015, yet the government kept up its anti-immigration xenophobia. It is against this backdrop that Orbán's government decided to tackle the crisis of care by foregrounding the problem of the demographic crisis at the cost of any other issues related to the problem of care. "We want more children, not migrants" said Hungary's prime minister as part of his re-election campaign in March 2018 (Erdély.ma 2018), and Hungarians found this call appealing. In 2018 Orbán won his third election victory and gained a qualifed majority in Parliament. The scene was set for the fnal development of the carefare regime. # Social Citizenship Claims in Carefare Regimes In her now classic account of the history of the Hungarian welfare state, Lynne Haney (2002) notes a shift around 1985 in the "architecture of need" that underpins the logic of social citizenship. Before the mid-1980s claims to the state socialist welfare state were most successfully made on the basis of maternity. State socialist social policies and centralized redistribution guaranteed that women with the same number of children received the same amount and types of subsidies regardless of other circumstances, such as need or work status. In my previous work I argued, in agreement with Haney, that women during state socialism were constructed as a "corporate" group, with specifc skills and unique contributions to society, typically maternity. This guaranteed specifc rights and privileges, different from those of the group of men (Fodor 2003). But in the mid-1980s these "maternalist" principles changed toward what Haney calls "materialist" ones. She argues that instead of motherhood, material needs came to be seen as the basis of social citizenship: new measures were introduced that targeted the group newly defned as needy, state provisions were allocated on the basis of material need, evaluated by local rather than central governments and in a way that differentiated among women, rather than offering similar support to all in the same care work or parental category. Means tests were utilized and benefts favored those who had paid social insurance, although beneft levels differed according to other characteristics too, such as the length and type of previous employment or education. Overall, subsidies were cut back signifcantly, or they lost their real value in the context of infation and became subject to political struggles. "Child rearing was no longer considered a social responsibility deserving remuneration; women were no longer guaranteed compensation for their maternal labor; and claims to state assistance were no longer framed around one's contribution as a worker, mother, or family member … women would be recognized only as 'needy' individuals" (Haney 2002: 189). "Materialist" welfare principles are part of the logic of neoliberal statecraft; and neoliberal-leaning capitalism was understood in the early 1990s as the brightest possible future for the country. Notably, as distinct from membership in the corporate group of women (i.e., potential, present or past mothers), neoliberal citizenship rests on the notion of the individual. As Rose (1998: 165) put it, "The political subject is … an individual whose citizenship is manifested through the free exercise of personal choice amongst a variety of options". Individuals are expected to develop their human capacities in order to compete successfully on various markets and they must strive to rely on these markets to satisfy their needs. The state is of minimal importance, providing fnancial support only in cases of dire need. Anti-liberal Hungary moved away from this individual-based principle of social citizenship. In Orbán's "carefare" state claims to the state can again be made on the basis of membership in a community, specifcally the family and, indirectly, the nation. (This is not the only basis of claims making but certainly a new and very signifcant one.) In the words of László Kövér, the President of the Hungarian Parliament, "Who is a decent Hungarian citizen? Not someone who speaks Hungarian. It is someone who has 3–4 children, 9–12 grandchildren, they all speak Hungarian and are committed to the Hungarian nation" (László Kövér's speech in Gyergyószentmiklós, August 2019). In his interpretation, citizenship including social citizenship—is based on active membership in a fertile, populous family. Government policies have been duly transformed to refect this principle. A quick review of the regulations introduced between 2014 and 2020 makes clear the explicit goal of encouraging childbearing—as well as the additional requirement of parents' participation in paid work. I have enumerated the very long list of the main policies—both new and old—in the appendix. Column 1 identifes the basis of receiving the beneft. Note that the state has eliminated, shortened, cut or devalued all universal cash benefts paid to people as a citizenship right. As an example, see the backbone of family protection legislations since the mid-1960s, the fat rate threeyear parental leave allowance (line 3 in the table in the appendix). For the past several decades this has been the main social beneft available to new parents, which allowed mostly mothers to withdraw from paid work for up to three years after childbirth to raise children. Parents receive a lump sum payment, retain social insurance and the promise of their jobs back upon return. Parental leave is an extremely popular measure in Hungary, where the majority of the population is convinced that children do best if they are raised at home by their mothers until age 3 (Blaskó 2011). Only one government attempted to abolish the three-year leave as part of a broader austerity package, but the policy was soon reinstated. The actual sum the participating parent receives equals the minimum old age pension in Hungary, which has been set at 28,500 HUF (or less than 80 EUR) since 2008. At that time in 2008, the parental leave beneft represented 41% of the national minimum wage, while in 2020 it amounts to only 18% (and about 7% of the average wage). To counter any hope that this may be adjusted, the government additionally passed a regulation which essentially froze the value of the minimum pension/parental leave beneft at this level forever (Government decree 707/2020. (XII.30); Portfolió, 2021). At the same time a different type of parental leave beneft has been raised generously: the one that mothers with formal employment can claim for the frst six months of their leaves. The government has boosted the value and conditions of insurance-based parental leave options, while allowing the universal fat—rate beneft to devalue. To complement the parental leave allowance, the "family beneft" has served to support families raising children since before World War II. The regulations and eligibility criteria of the family beneft have changed several times over the years, but it has been a universal support scheme since 1998 (Spéder et al. 2020). However, its value has fuctuated signifcantly. At the end of the state socialist period in 1989 it represented 21% of the average wage per child, which was a signifcant contribution to the family budget. It was devalued after 1990, then adjusted during the reign of the Socialist government in 2008. Since then, neither the Socialist government in power until 2010, nor Orbán who took over in the spring of that year, has changed the amount of support allocated per child. This means that in 2019 the beneft for a single child amounted to less than 5% of the average net wage (author's calculations based on data from Central Statistical Offce, and Jarvis and Miklewright 1992). Both major universal benefts—the three-year parental allowance and the family beneft—which people receive without consideration of class or work status have lost a signifcant portion of their values and the government has announced that it has no plans to change this. Several other types of social provisions met the same fate (Scharle and Szikra 2015). Successful claims on the state are nowadays made on the basis of signifcant care work combined with some employment history. Claim makers are typically "families" (defned as heterosexual married couples with children and employed in the formal economy) rather than individuals. The frst and most signifcant of new family benefts is the earned income tax credit, which was re-introduced in 2011. On its offcial webpage, the government describes it as refecting two basic values: "work and childcare done in addition to paid work". Note the emphasis on the combination of care work and paid work—this is certainly not a traditional "back to the kitchen" ideology! Working parents can claim a portion of their taxes back, which in 2021 could yield, at the maximum 33,000 HUF per month (about 100 EUR) per child if a family has at least three children. The tax break is signifcantly smaller per child if the family has fewer children, which is not surprising since the government has an openly pronatalist agenda and the measure is meant to encourage childbirth. The goal is in fact noble: demographers had long argued that Hungarians wanted more children than they would actually end up giving birth to, so the government claimed that it sought to redress this problem and enable families to have as many babies as they desired (Kapitány et al. 2019). But earned income tax credits can only be claimed by those in formal employment, and informal work arrangements are widespread in Hungary (Hegedűs 2020). In addition, the total family income from formal employment must exceed a certain level, otherwise parts of the tax break are lost. In 2019, families had to have a joint income of at least 330,000 HUF, which was about 10% lower than the average gross wage for full-time work for one person. Recent studies, however, show that about 40% of employees are registered at the minimum wage in Hungary, although they may or may not receive additional income "under the table" (Hornyák 2019). The minimum wage was 149,000 HUF per months in 2019, so even two parents working full time on the minimum wage were not eligible for the full sum. If parents divorced—and about a third of marriages end up in divorce in their frst 15 years (Makay and Szabó 2018), only one party can claim the tax credit, typically mothers, whose income alone may not be enough to receive the full sum. Nevertheless, a signifcant number of families can and do utilize these earned income tax credits—the precise number is simply not available. Those without employment or formal employment are not eligible. Following a similar logic, working mothers of four or more children do not pay income tax—a beneft that is tied to both paid work in the formal economy and signifcant care work responsibilities. In addition to the tax credit, married couples can take a variety of loans which do not have to be paid back if they give birth to the requisite number of children. The centerpiece is a 10 million HUF loan (about 27,000 EUR, introduced in 2019) for couples who plan to have children. The loan can be used for any purpose and a portion is forgiven after the second child. It turns into a non-repayable grant once the third child is born. Couples do not pay interest in the frst fve years, or at all once they have their frst child. Signifcantly, this is a loan, which can only be taken if at least someone from the family has a three-year work history, a sizeable formal income, and if the couple is married. Its value, as must be obvious from the above, far exceeds that of the fat rate family beneft. A second set of provisions helps "families" buy, build or renovate their homes. If married couples promise to have children, they become eligible for a grant to be spent on real estate, the size of which is dependent on the number of children they have, or promise to have, and the qualities of the home. There are additional subsidized loan opportunities, mortgage reduction and a handful of other variations on this beneft. The main point is that they are all tied to employment, being married if the couple has no children yet, age (she has to be under 40), and the couple must have signifcant resources of their own because the state subsidies are not enough in and of themselves to buy/build or even renovate any real estate. Interestingly, there is a special provision for those who want to build a multigenerational home, which the government encourages and offers special subsidies for. Grandparents are encouraged to be involved in care work via other means as well: they can take parental leave instead of the birth parents of the child. In that case, however, not only the grandparent but both parents of the child must be insured, thus working for wages. All combined these provisions target married couples with some formal employment and savings of their own, who have or plan to have at least two or more children. The sum they can claim from the state is sizeable, while provisions available outside this social category are lower and disappearing. In Hungary's state socialist "maternalist" welfare regime, mothers of all social categories received similar benefts regardless of employment, marital status or social class. Social citizenship claims in Hungary's carefare regime, on the other hand, are conditioned on a combination of criteria related both to formal employment and to unpaid care work done within a married couple family. # Selective Disinvestment and Churchification What Peck and Theodore (2012: 179) call "the prosaic and frequently tawdry practice of [neoliberal] deregulatory statecraft" typically involves disinvestment in social protection measures, and the decentralization and commodifcation of formerly state provided services. David Harvey (2015) goes even further to claim that neoliberal states have become active agents in capitalist "accumulation by dispossession" and under the pretext of deregulation seek to create a favorable climate for business interests, protect the integrity of fnancial institutions over community interests, and redistribute wealth in a way to keep large segments of the population impoverished and corporate capitalist greed satisfed(Harvey 2015). In this context it is the "overgrown penal state", which keeps the poor under control, and thus neoliberalism "entails not the dismantling but the reengineering of the state" (Wacquant 2012: 6), specifcally "marketconforming" state crafting. This is a political rather than an economic project, in which the state re-regulates the economy, commodifes existing services, imposes disciplinary social policy to replace both welfare and workfare systems, and uses penal policy and the discourse of individual responsibility to keep people in line (ibid.). It is in this context that the crisis of care becomes more apparent than ever. The exclusive emphasis on proft leads to a devaluation of reproduction, and reduces support for the birthing and raising of children, caring for the sick and the elderly, and maintaining social ties which hold together families and communities (Fraser 2016; Isaksen et al. 2008). In good neoliberal fashion, the Hungarian state has also withdrawn funds from social protection in the past decade. In 2010, Hungary dedicated 17.4% of its GDP to social protection expenditure, which was reduced over the years to 13.3% by 2018, while the EU-27 average remained stable at roughly 19% (Eurostat 2019a). Indeed, as Prime Minister Orbán pointed out, the aim of the Hungarian government was to transform Hungary from a welfare to a workfare state, which meant reducing need-based provisions to the bare minimum, and securing workplaces to those willing and able. Following this logic, the Orbán government invested in developing a public works program which soon grew to be one of the largest in the world, and, as noted above, cut cash payments of all varieties, replacing some with benefts in kind. The length and value of the unemployment benefts was decimated, access to long-term sick pay became cumbersome and the value of a variety of social provisions to the needy was devalued. OECD statistics confrm that the proportion of the country's GDP spent on cash transfers decreased signifcantly while inkind services have remained stable since 2010 (OECD 2019). Up to this point, a familiar picture is presented: parts of the world exposed to global neoliberal economic policies and the structural adjustment requirements of international fnancial organizations often follow these patterns. In fact, this strategy is not altogether different from what several rather liberal Hungarian governments had pursued in years prior to Orbán's accession to power. On closer inspection, however, one important distinction emerges. In 2019, Hungary and Germany were recorded as the two countries within the EU which spent the largest share of all social protection costs, 12%, on a specifc function, namely "family protection". When the costs of earned income tax credit are added, Hungary is one of the world leaders in this regard (Makay 2018). To be clear, family protection is euphemism for pronatalist policies offering incentives to heterosexual Hungarian families to have more children. So while spending on other areas of social protection, such as welfare and unemployment declined, "families" have been targeted with generous support. (The quotation mark is a reminder that only one specifc form of household is considered a family: heterosexual couples with children and with some form of paid employment.) The government has been funding this specifc area of reproduction generously. Although this is just one segment of care work, it is an important one, and one which demonstrates that instead of neoliberal state retrenchment, in Hungary we are witnessing the reorganization of state capacity in line with specifc political goals. Critiques of neoliberal governance note with alarm the increase in deregulation of state services in western economies. A curious alternative has been emerging in Hungary. First, certain state services have in fact expanded: the government has been building nursery schools and has increased access to state provided childcare. Although a small number of private providers are also on the market, their share is insignifcant: fewer than 10% of children spend time in paid day care (KSH 2019c). In other areas, the state is in fact deregulating, but favors only a specifc type of provider: a handful of trusted churches. In this case the goal is not to cut costs, as the state funds these services more generously than it does its own institutions. Instead, the goal is to build political loyalty for present and future generations. Churches have played a growing role in social services in Hungary since the collapse of state socialism, but their participation has increased exponentially in the past decade. By 2020, churches ran about 25% of homes for the elderly and the disabled, provided 45% of all basic social services, and 60% of all child protection services (Magyar Nemzet 2020, quoting the Minister for Social Affairs). Care in these institutions is not paid for by donations from members of the church community. Instead, the Hungarian state allocates resources to a small number of established churches to provide the same service as the state or civil organizations do. Only churches receive more funding per capita than a state or non-governmental provider would and they are not obligated to spend all of the money on the actual service in question. By a recent decree, the largest churches also receive the property rights of the institutions they run, cementing their role in the feld and allowing them space for independent economic activities. The lack of separation of the church and the state is especially poignant in primary and secondary education, where religious schools have multiplied at the cost of funding good quality secular public institutions. In 2001, fewer than 5% of children attended schools run by churches. In 2019, 15% of primary school students and 25% of secondary school children did so. While in western liberal democracies engaging forproft and non-proft providers allows states to control and cut costs, in Hungary state services are not commodifed or marketized but churchifed: increasingly overseen by politically and ideologically loyal religious organizations, which preach a specifc ideology, and support the sustenance and reproduction of an anti-liberal political order. # Women Do Care Work in "Families" "I would like to make a deal with Hungarian women, Hungarian ladies, about the future and their role in it as well as the new opportunities the government could offer", suggested Prime Minister Viktor Orbán in a radio address on the national radio channel in April 2018, soon after his third election victory. His offer clearly refects a key principle of the new pronatalist policies he was referring to: having children is women's job, women's decision, they are the ones responsible. Since women are expected to do the work of birthing and caring for children, the prime minister's offer addresses women and women alone. The third feature of carefare regimes is the unashamedly unequal distribution of care work and the emphasis on the household as the location for care. The literature on the de- and re-familialization of care is extensive (Mahon 2002; Morgan and Zippel 2003). Familialism denotes policies, which encourage care, especially childcare, to be carried out within the family. The opposite of the concept is de-familialization, that is, when policies encourage the outsourcing of care and thus open up space for women's successful participation in paid work, the two-earner family (Javornik 2014). Tendencies of re-familialization have been observed in most post-communist societies and several typologies exist to describe different types of policy packages (Fodor et al. 2002; Haney 2003; Javornik 2014; Rat and Szikra 2018; Saxonberg and Sirovatka 2006; Szelewa and Polakowski 2008; Szikra and Szelewa 2010). These studies typically take into account two large sets of policies—parental leave and the availability of childcare—and show how various combinations offer different options for women. Some encourage them to do care work at home, others to work for wages and send children to childcare institutions, while yet others allow families to choose between these two options. My argument is that in Hungary's carefare regime women are assigned care work and care is primarily relegated to the home, and simultaneously, they are expected to work for wages full time. The confict between reproductive work and work is solved via women's increased work burden and exploitation. Hungary's recent "family protection" policies aim to increase the number of births. While most demographers agree that they are unlikely to raise the total fertility rate to the point of replacement, the policies could, at least temporarily, increase the birth rate in at least some segments of society (Spéder et al. 2020). Indeed, following the introduction of the tax credit-based benefts, in 2014 the number of births increased in Hungary, and after a drop, picked up again in 2020. Birth rates have grown fastest in the poorer regions of the country, while the decline in births continued in the capital of Budapest (KSH 2020). As the number of births per woman increased between 2010 and 2020 from a low of 1.33 to 1.5 and is likely to grow further, families' reproductive burden is also expected to become heavier. And, as time budget surveys indicate, the brunt of this extra work will most likely be shouldered by women. In 2010, the year for which the most recent data are available, mothers of two children, living with their spouses, spent 96 minutes a day on childcare (while their spouses also dedicated 37 minutes to this task). But in families with three children, mothers spent an additional 82 minutes more a day on childcare, over three hours altogether, while fathers of three children only did 15 minutes more than fathers of two. The extra care burden of another child is clearly carried by women (Falussy and Harcsa 2000)and even if we count the gender difference in the length of paid work, mothers had about an hour less free time per day than fathers in 2010 (KSH 2012). Mothers' burden is likely to increase especially as intensive mothering is becoming more popular in Hungary too. In a small survey we conducted in 2020 during the COVID-19 pandemic, we found that while all mothers increased their care work during the lockdown months more than fathers did, educated urban mothers' workload grew about four times more than the average (Fodor et al. 2020). In general women at all levels of education spent more time than fathers helping their children with school work and educated women especially seemed to have internalized the expectation of intensive mothering and the notion that they were responsible for making sure their children were not left behind (Geambasu et al. 2021). More children thus mean more work, and that work will most likely be women's responsibility. While a great deal of propaganda is dedicated to encouraging women to have more children, no mention is made of men's role in carrying at least some of the care burden. The Hungarian government has made several generous adjustments to parental leave benefts which are almost exclusively used by women. However, it did not increase the length of paternity leave from fve working days, which is signifcantly lower than the EU average of 12.5 days. Welfare typologies usually consider the division of labor with a nuclear family—type household in mind (although see Utrata 2015). In Hungary, familialization has traditionally included the mobilization of grandparents for care work as well. Although the proportion of multi-family households has declined in Hungary over the past 50 years, still about 23% of women and 18% of men over 65 live with their offspring (Monostori and Gresits 2018). Most grandparents are heavily involved in the care of children: in 2016, two-thirds of 55–79-year-olds participated in this activity. The younger, healthier and more educated they were, the more likely it was that they helped out (ibid.): an impressive 80% of college-educated grandparents looked after small children in Hungary in 2016. Interestingly, in this age group there was little gender difference: grandfathers were almost as likely to take care of children as grandmothers (ibid.). Building on this tradition of multigenerational care, the government has created fnancial incentives for grandparents to take parental leave instead of their children, and is offering special support to those who seek to build multigenerational households and to retain care responsibilities within it. In return, the government enshrined in the Constitution of 2011 the obligation that children take care of their elderly parents in need. This, obviously, is another glaring instance where care is familialized, although not via incentives but legal decrees. In conclusion, in Hungary's carefare regime mothers are primarily responsible for care work in the home. A very small number of families can afford to rely on paid help, although more take advantage of grandparents' availability. The government's pronatalist policies have already resulted in the birth of more babies and this is likely to continue. There is no public mention of the fact that having more children will surely increase women's care work load. # Beyond "back to the kitchen": Women as Wage Workers The fourth feature to note in Hungary's carefare regime is the one least discussed in the literature: the necessity for women to be engaged in paid work in addition to producing additional Hungarians for their families and the nation. As I showed above, benefts claimed on the basis of care responsibilities are also tied to employment history, either explicitly or because they require a level of income which is only achievable by two earners. In addition, specifc targeted regulations directly encourage women's return to work. For example, in 2014 a change in parental allowance was introduced, which means that women can now keep receiving the allowance even if they go back to work and, unlike before, they can work full time. Employers too have long had some incentives to hire women with small children through a reduction in taxes on labor. This distinguishes Hungary's anti-liberal carefare regime from European conservative welfare arrangements, such as, for example, in Germany or Austria (Shire and Nemoto 2020). In this carefare regime the "male breadwinner" model or the notion of the family wage are not ideals to be followed, quite the opposite. In Hungary, women have long been permanent participants in the labor market, and their wages have been essential for the family budget. At the same time, women are also responsible for care work in the home: they drop out of the labor market for lengthy periods after childbirth and dedicate signifcantly more time than their spouses to the daily chore of raising children and doing other types of care work. This necessarily limits their opportunities in paid work, puts them in a precarious position in the labor market, and occasionally forces them to accept trade-offs between wages and the ability to meet their reproductive responsibilities (Mandel and Semoyonov 2006; Petit and Hook 2009). Two issues clearly differentiate carefare regimes from others which encourage a dual wage earner model. First, the stated goal of achieving gender equality in the labor market is missing in Hungary as are policies which require that employers, including state employers, guarantee transparent and reliable work–life balance measures. Second and related, instead of equality legislation or workers' representation, women's work is "sentimentalized": women are constructed in offcial political discourse as primarily carers, even in the workplace. Care work is devalued and, as elsewhere, it is understood as part of women's true feminine identity, not as part of their job description (England 2005; Hochschield 1983). The combination of these two factors—sentimentalization and the lack of gender equality/care work reconciliation measures—result in an increase in the "motherhood penalty", that is, the disadvantages mothers suffer at work, and exacerbates workplace gender inequality, especially at the bottom of the social hierarchy. It leads to the emergence of an underclass of women workers, who may have the opportunity to be earning an income in humiliatingly underpaid jobs but must struggle on a daily basis to hold on to their positions and manage their care responsibilities simultaneously (Gregor and Kováts 2018, 2019). I will start with a discussion of this latter phenomenon. #### An Underclass of Working Women Paid work opportunities soared after 2015 as the Hungarian economy, partly fueled by payments from the European Union's structural funds, picked up speed in the aftermath of the economic recession. In 2019, the unemployment rate stood below 4% and remained under 5% even during COVID-ridden 2020. As Fig. 2.1 shows, both men and women were able to fnd work and while in the early 2010s Hungarian women's labor market participation rate counted as one of the lowest within the EU, by 2020 it had climbed to average levels (Eurostat 2019b). Two points are important here. First, women's employment growth seems to be slowing after 2012: the gender gap in employment started to widen slowly but perceptibly. Indeed, Eurostat data indicates that, compared to men, women in Hungary are less likely to be able to transition from unemployment to employment. This is true for most countries but the gap in Hungary was fve percentage points in 2019 (as well as in several prior years), which is twice the EU-27 average, and higher than in other CEE countries (Eurostat 2019c). The COVID pandemic further increased the difference in the number of employed men and women (KSH 2021). Second, the gender gap in access to paid work is particularly large among those with lower levels of education, that is, at the bottom of the Fig. 2.1 Changes in men's and women's employment rate, 15–64 year-olds. (Source: Eurostat 2019c) occupational hierarchy and there are signifcantly more women than men in this group. But the number of employed women in this category has been growing. While in 2011 only about 31% of women with elementary education were working for wages, this percentage increased to 46% by 2019—a close to 50% growth, larger in absolute numbers and percentage terms than in any other educational groups (ibid.). This is the underclass of women workers I mentioned previously. Working for wages is not all bad. Employment opened up new, if rather limited, fnancial opportunities for women at the bottom of the occupational hierarchy. It, however, also exposed them to more gender inequality, more work and harsher exploitation. Women's wages are lower than men's in Hungary and women in the lower educational category experience roughly the same wage gap as the national average of 16% (Eurostat 2019d). Lower wages are less likely to help pull someone out of poverty and this is what we see in Fig. 2.2. Women's at-work poverty risk had been lower than men's until 2015, after which it started to exceed men's. Working women's risk of poverty doubled from 4.6% in 2010 to 8.7% by 2019 and exceeded men's which stood at 8.1% in 2019 (see Fig. 2.2, based on Eurostat 2019e). In other words, parallel to the mass entry of women into low level jobs, the risk of in-work Fig. 2.2 At-work poverty risk by gender. (Source: Eurostat 2019e) poverty increased, suggesting that women's wages are less likely to move their and their families' living conditions above the poverty line than men's. Note that the women who are classifed as poor in this chart may have been poor before 2016 as well, but they were not included in these statistics as they did not have paid employment. In the middle of the 2010s they started to join the ranks of underpaid, precarious workers. This underclass of women is heavily overrepresented among workfare workers. In an effort to eliminate what Prime Minister Orbán called a "welfare society", the Hungarian government boosted workfare programs to the point where close to 200,000 people participated at the peak in 2016. Workfare participants get paid a fraction of the minimum wage and typically work in menial jobs, which do not enhance their labor market chances (Cseres-Gergely and Molnár 2014). Studies suggest that in rural areas workfare opportunities are often allocated in exchange for political favors (Róna et al. 2020). Yet, workfare arrangements are popular because the alternatives are even worse. As noted before, the government has all but eliminated other forms of support for those who lost their jobs. And workfare has other advantages as well: it is a form of formal employment in reasonably regulated, typically single-shift, and occasionally part-time, conditions. These are job characteristics which are not easily available to low skilled workers. Importantly, given that workfare workers are in the formal economy, they also become eligible for tax benefts for children. As a result of all these and other labor market related factors, workfare programs have become feminized in the past years (Fekete 2021). In summary, more women have been working for wages in the Hungarian economy in the late 2010s than at any time since the transition from state socialism but job growth was largest at the bottom of the occupational hierarchy, among the unskilled and also among those who work in the vastly underpaid government workfare programs. In addition to the opportunity to claim child benefts, women also occasionally choose workfare jobs over other types of employment because regular employment shift work, informal work without contracts, the requirement to do unannounced overtime, the lack of control over the timing of the work period and the diffculty in fnding part-time options—make it hard for women to reconcile childcare and paid work duties. In a recent study Dorottya Fekete (2021) asked workfare workers with children about their motivations. She found that what they appreciated most were the family benefts they gained access to, as well as the more family friendly work option of the possibility to work part time in single day time shifts. The COVID-19 pandemic of 2020–2021 further exposed the vulnerabilities of women's employment: in Hungary, as in many other countries around the world, more women lost their jobs than men. According to aggregate data from the Hungarian Statistical Offce, between the end of 2019 and 2020 (the last quarter in each year), the employment rate of men aged between 15 and 64 in fact increased by 0.2% (even though the actual number of those employed declined somewhat), while women's rate decreased by 0.4%. Job loss was more pronounced among those with less education, and among those employed in public works programs. However, even among the college-educated population where the number of the employed in fact continued to grow during the pandemic, men did signifcantly better than women, widening the employment gap within this group (KSH 2021). This is most likely explained by the extremely unequal division of care work which prevailed in this social stratum (Fodor et al. 2020). The "motherhood penalty", as this phenomenon is called, is the topic of the next section. #### Inequalities Among All: The Motherhood Penalty Hungary has the most generous set of parental leave and family benefts policies in Europe, yet also the fewest and most stingy work–life balance measures. Together with the requirement for mothers to be working for wages, this creates obvious inequalities. Indeed, as my calculations based on the Survey of Income and Living Conditions (EU SILC) data show, mothers of children under 16 years of age, net of other characteristics such as age, work experience, education, number of subordinates, work hours, marital status and whether or not they live in a multigenerational household—make less money than women without children. Fathers, in fact, experience a bonus over non-fathers. This was true in 2010, and the coeffcient had increased statistically signifcantly by 2017: the motherhood penalty had grown.2 This is not surprising if we consider the dearth of policies aimed at supporting those with care responsibilities in the labor market. Researchers often talk about the reduction in labor rights during the Orbán era: strike <sup>2</sup>The dependent variable was the log of income, individual variables are listed in the text. The variable of interest is the interaction between parental status and gender and I ran joint models for the two years with interaction terms to make sure that the change was signifcant. More information is available upon request. laws have been changed, overtime payment was reregulated to favor employers, unions were weakened and so on (Scheiring 2020). Rather less discussion has been dedicated to the extremely weak rights workers with care responsibilities have (for exceptions, see Gregor and Kováts 2019; Juhász 2012). Work hours in Hungary, as in other post-state-socialist societies, are higher than in most EU member states. Yet an extremely small number of people can work part time, only 4% in Hungary, one of the lowest rates in the EU. This is partly women's choice: wages are so low that a part-time salary is not enough to maintain a household. At the same time, even if they want to, it is diffcult for women to negotiate part-time options with their employers (Fodor and Glass 2018; Glass and Fodor 2011). According to data from EIGE, the European Institute for Gender Equality, Hungarians are less likely to be able to set their own work hours than other EU states, and women, in particular, claim that they have no fexibility in this regard. In comparison to citizens of other EU countries Hungarians are less able than to adapt their work hours to external needs, they are less likely to be able to determine their own work hours and face diffculties when they need to take an hour or so off for personal reasons during the workday (EIGE 2019). None of this is surprising as no consistent government incentive exists to prompt companies to enact work–life balance policies. Quite the opposite. In 2014, a new regulation was passed, which required that state administrators in one of the largest ministries (state department) and its local administrative offces work ten hours a day from 7:30 to 17:30. At the time, the minister in charge did not mince words when he claimed that "Our job has a beginning but it has no end, so this state department is unfortunately not a family friendly workplace" (János Lázár's speech in November 2014). The claim was preposterous, not only because of its lack of acknowledgment of the care work burden of his mostly female workforce, but also because Lázár ignored national legislation about compulsory work hours. Nevertheless, a ten-hour workday was instituted and was in effect for four years before it was rescinded, just as abruptly as it had been implemented. Note that this rule came to be applied to several other state institutions as well, all offering the type of pink-collar work that is typically understood as representing an opportunity for mothers to reconcile work and care responsibilities elsewhere. The Hungarian government did not exactly show a good example to privately owned companies, which clearly demonstrates its lack of dedication, attention and consideration to those with care responsibilities. Even during the pandemic, a relatively low number of people could work from home in Hungary, although more women did than men (Eurofound 2020). An OECD survey showed that, in general, Hungarian workplaces are not fexible in terms of work location: in 2015 fewer than 20% of mothers could do their jobs from a home offce at least once during the year, placing Hungary into the bottom third of this distribution within Europe (OECD 2019). This is so even though quantitative and qualitative surveys demonstrate that women with care responsibilities would very much prefer to have this option available (Gregor and Kováts 2018). Institutional childcare eases some of women's care burden. Kindergarten places for children over three years of age are fairly easily accessible in Hungary, with the exception of areas far from larger settlements. This is not the case for nurseries; although Orbán's government has dutifully utilized earmarked EU resources to build more child care institutions. This is refected in a moderate increase in attendance over the past decade. In 2010, over 90% of children under three years did not attend any formal childcare institution, while in 2019 only 83% did not, which is still far from the EU average of 35% (Eurostat 2019f). In addition, childcare facilities are rather infexible in opening hours: most close at 5 pm and there is little leeway for extra hours or a different schedule. This constrains parents', mostly mothers', work time options. Even when work–life policies do exist, their implementation is not necessarily automatic: women feel that they are asking for a personal favor. In a research project conducted with Christy Glass among professional mothers in Hungary we found that they do not consider part-time options (although legally guaranteed for women returning to work with children under three years) or parental leave policies (also enshrined in law) as true entitlements. Instead, they had to negotiate the terms of their leave, as well as their return, and were dependent on the goodwill of their supervisors. As a result, some women managed to get an arrangement that was acceptable and allowed them to balance work and family, while those whose supervisors were less understanding did not; in some cases this resulted in major breaks or shifts in women's careers (Fodor and Glass 2018). Personal connections and the importance of social capital are deeply embedded in Hungarian social institutions and history, which is one of the reasons why the women we interviewed did not fnd the necessity to negotiate legally mandated rights problematic. # Instead of Gender Equality Policy: Sentimentalization Through its anti-gender discourse and conspiracy theories about the EU's gender lobby, the government absolved itself of responsibilities regarding women's equal opportunities in the labor market. The quote at the beginning of the chapter from the Minister of Family Affairs reinforces this. Katalin Novak suggests that women should not be seeking equal wages to men; they should be content with the opportunity to be "real" women, to give birth and to take care of others. She is not alone in her open denial of the principle of equality. The President of the Hungarian Parliament argued this in 2019: We should not overemphasize equality as that would mean the abolition of genders and in the end the rejection of femininity and women's virtues. (LaszlóKövér, in FICSAK 2019) In other words, equality would threaten women's identity as women; femininity is essentially the opposite of gender equality. It is in this spirit that the Hungarian government has ignored EU-wide gender equality action plans, road maps and policy recommendations. A national-level round-table involving experts, NGOs and government offcials on gender issues was discontinued, the section of the ministry which dealt with gender equality closed down, and the government frst de-funded and then closed the Equal Opportunity Commission which had been designed to oversee problems related to gender-based discrimination, despite the fact that it is an EU requirement to have an EOC in place. The Hungarian Parliament has refused to ratify the Istanbul Convention on the elimination of violence against women, including domestic violence, and the Prime Minister has threatened to veto any EU regulation containing the word "gender", including the Action Plan for Gender Equality III. I argue that instead of striving toward gender equality, the government has started to sentimentalize women's care work and closely confound it with women's identity as women. To illustrate I analyze quotations from a government funded publication in which men wrote laudations to women on the occasion of the International Day of Women in 2019. These were collected in a booklet and published by an organization called the Organization of the Club of Young Families (FICSAK 2019). (More on the publication in the methodological section of Chap. 1.) Practically each one of the quotes from top-level politicians published in the booklet mentions how women excel in care work, both in the home and in the workplace. As the Minister for Defense argues: "We think of women as the weaker sex, but they represent real spiritual power. They represent persistence, selfess dedication and love and all they expect in return is respect, attention, appreciation, love and kindness." Or in the words of the Minister of Human Affairs: "You [women] are caring, attentive, empathetic, beautiful. You give birth to children, you are the heart and soul of families." To quote a state secretary in the same ministry, "We need women to make our institutions, our communities, our families accepting, warm and caring". In all these cases womanhood is associated exclusively with caring and related attributes including kindness, devotion, gentleness, understanding and so on, and the production of these feelings in various communities is assigned to women and women alone. At the extreme, here is an example from a deputy minister who explicitly identifed women's role as being men's primary support mechanisms. He wrote, "It is women who help hold the World together, and who we, men, can rely on day after day in our work, at home, in our communities". The quotes associate women with acceptance, warmth, providing care and support, and none mention productivity, creativity or intelligence, even within these attributes. In the workplace too, women represent the very same qualities. "I want to thank my own female colleagues … that they always suggest the possibility of a compromise not only in the family but in the workplace as well" chimes in a state secretary from the Ministry of Finance. Importantly, several men acknowledge the fact that it is hard work to be caring for a family and working for wages simultaneously. Women are expected to be overworked, and it is considered to be women's special skill to tolerate this. As a state secretary put it, "Only you [women] are able to do this: be a mother and a wife and at the same time do well in your job as well"; or in the words of another high-level male politician: "We often forget the many challenges women must face in our world today. They have to work for wages and must be perfect wives, mothers, problem solvers." "Nothing compares to women's performance. In addition to their visible—paid work … [they also take care of their family]. … This is hard work. It is a calling, rather than a simple job yet they do it smiling, without complaint, naturally" (Minister of Finance). Although the men acknowledge the exceptionally hard work women do, note the multiple references to a calling, rather than a form of skill or hardship that requires or warrants compensation. "Being a woman is more than a simple task. It is a calling" repeats one of the state secretaries of the Parliament. Women are due respect and appreciation but not tangible rewards. The politicians here sentimentalize women's work: they elevate it to the level of a calling, where fnancial incentives and rewards seem meaningless. Let us end this section with the words of Hungary's young Minister of Finance, who seems to be familiar with the term "invisible work" and acknowledges that women do most of it, both in the family and in the workplace. But "they do this out of the kindness of their hearts without expecting remuneration of any form, simply because they consider it the right thing to do". In Hungary's carefare regime, femininity is closely tied to selfess care work within and outside the family setting. Men take part at their pleasure, but care is women's primary responsibility. The work is much appreciated, it is considered important and socially valuable, but not remunerable. Care work is sentimentalized rather than commercialized. # Conclusion I have argued that a new response is emerging to the crisis of care in Hungary. The policy direction I call "carefare" aims to eliminate the contradiction between intensifying production and the need for reproduction by piling additional work burden on the shoulders of women and taming their possible reluctance by discursively connecting femininity to care work. As several researchers have pointed out, the role of the state in managing the economy, society and redistribution is extremely important in antiliberal Hungary (Magyar 2016; Scheiring 2020). From this position, the government has made good political use of Hungarians' long-standing anxieties about the disappearance of their bloodline and the death of the national culture due, allegedly, to women's reluctance to reproduce. Demographic revival was placed on the agenda of the Orbán government immediately upon gaining power in 2010 and it became its true focal point after 2015. As a consequence, over the past decade, the Parliament has passed a whole slew of policy measures to encourage births, specifcally to encourage births to heterosexual, married, working families. In the process, the state has created a carefare regime: encouraged women to have children, and do most of the associated care work, while simultaneously constructing them as second-class, female workers on the paid labor market. Claims to social citizenship are now most successfully made on the basis of parental and work status combined. This move toward a carefare regime has a number of consequences that are already visible: the most important is the growing underclass of female workers who work for extremely low wages in exchange for being able to maintain their labor market status while also taking care of their dependents. I also noted the increased work burden that a higher level of reproduction—within the context of the unequal distribution of care work—means for women. At the same time, carefare provides political capital to the Orbán regime: it functions as a mechanism to lessen the pain of increasing social inequalities. Especially among the lower middle classes, women's extra work may buy households out of poverty, or at least allow them a degree of upward mobility, even though this may be limited or fragile. Economic growth has resulted in a signifcant increase in class inequalities in Hungary, although some resources have trickled down to certain groups of the population, especially to families with children with some attachment to the formal labor market. They are some of the government's most loyal supporters and the voters who brought FIDESZ into power (Róna et al. 2020). Their families are doing better fnancially under the FIDESZ regime, thanks, in part, to the wide range of "family protection" measures available to them in the form of loans and government grants. The most positive development has been that the poverty rate of children has declined rapidly during the last few years in Hungary. In 2011 the poverty risk of a family with three children was a shocking 35%, but now it stands at 11.4%, one of the lowest in the European Union (Eurostat 2020). The same pattern is visible among families with fewer children, bringing the poverty risk of children down to levels below the EU average (ibid.). At the same time, however, the poverty risks of single people, of the elderly, especially elderly women, have all skyrocketed: they have been left out of the government provided windfall. While the reduction of child poverty is laudable, it should be noted that less than half of all households have children, fewer than a quarter have two or more children, and an increasing number of people are living alone. For them the carefare regime has little to offer. In conclusion, carefare regimes come with political benefts to antiliberal governments. Carefare eases some of the social tension which results from a rapid increase in social inequalities, obvious corruption and cronyism. By redistributing a sizeable, but still relatively small, amount of resources to a select group of "deserving" families, by increasing their social mobility chances, even if to a signifcantly lesser degree than that of families at the top of the social hierarchy, the government buys the loyalty of an important constituency. What we must not forget is that this is happening on the back of women, on the condition of women's increased contribution to care work and compromise in work options. In addition, upward mobility, even among those most favored by government policy, may be fragile. Over a third of all marriages end in divorce3 in Hungary and the small steps toward upward mobility may fall away quickly as divorcing couples have to share responsibilities for mortgages and loans, or when the promised number of children do not arrive, or as the job market ebbs and family income dwindles to the point where meeting interest payments becomes problematic. In this context women's sacrifce will be even more starkly visible. # References Fekete, Dorottya. 2021. 'Nekem ez most jó a kislány végett': Női tapasztalatok a közfoglalkoztatási programról. [This is good for me now because of my daugh- 3Of couples who married in 2000, over a third got divorced within 15 years (Makay and Szabó 2018). ter: women's experience in public works programs]. Szociológiai Szemle 30 (3): 70–95. ———. 2003. Welfare Reform with a Familial Face Reconstituting State and Domestic Relations in Post-Socialist Eastern Europe. In Families of a New World, ed. Lynne Haney and Lisa Pollard, 159–178. New York, London: Routledge. List of data and media sources ———. 2018b. Life Expectancy by Age and Sex. https://ec.europa.eu/eurostat/ databrowser/view/demo\_fnd/default/table?lang=en. Accessed 12 Feb 2021. ———. 2019a. Government Expenditure by Function. https://ec.europa.eu/ eurostat/databrowser/view/gov\_10a\_exp/default/table?lang=en. Accessed 12 Feb 2021. ———. 2019b. Employment and Activity by Sex and Age. https://ec.europa.eu/ eurostat/databrowser/view/lfsi\_emp\_a/default/table?lang=en. Accessed 14 Feb 2021. ———. 2019c. Labor Market Transition – Annual Data. https://ec.europa.eu/ eurostat/databrowser/view/LFSI\_LONG\_A\_\_custom\_564914/default/ table?lang=en. Accessed 14 Feb 2021. ———. 2019d. Mean Annual Earning by Sex, Economic Activity and Educational Attainment. https://ec.europa.eu/eurostat/databrowser/view/earn\_ ses18\_30/default/table?lang=en. Accessed 13 Feb 2021. ———. 2019e. In-Work at-Risk-of-Poverty Rate by Age and Sex. https://ec. europa.eu/eurostat/databrowser/view/ILC\_IW01\_\_custom\_565060/ default/table?lang=en. Accessed 14 Feb 2021. ———. 2019f. Children in Formal Childcare or Education by Age Group and Duration. https://ec.europa.eu/eurostat/databrowser/view/ILC\_CAIND FORMAL\_\_custom\_490064/default/table?lang=en. Accessed 14 Feb 2021. ———. 2020. At-Risk-of-Poverty Rate by Poverty Threshold and Household Type. https://ec.europa.eu/eurostat/databrowser/view/ilc\_li03/default/ table?lang=en. Accessed 14 Feb 2021. ———. 2019a. Népesség, Népmozgalom 1900–. Teljestermékenységiarány, élveszületések, terhességmegszakítások, halálozás. Budapest: Központi Statisztikai Hivatal. https://www.ksh.hu/docs/hun/xstadat/xstadat\_hosszu/h\_wdsd001b.html. Accessed 10 Feb 2021. ———. 2020. Élveszületések, 2019, 2020. Budapest: KözpontiStatisztikaiHivatal. https://www.ksh.hu/docs/hun/xstadat/xstadat\_evkozi/e\_wns001.html. Accessed 12 Feb 2021. ———. 2021. A foglalkoztatottak száma legmagasabb iskolai végzettségük szerint, nemenként. https://www.ksh.hu/docs/hun/xstadat/xstadat\_evkozi/e\_ qlf007.html. Accessed 12 Feb 2021. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. CHAPTER 3 # Fostering in a Carefare Regime Abstract This chapter is about foster parents and their work. Recent changes in the regulation of foster care illustrate the formation and operation of a carefare regime: the transformation of state policies and services and the integration of foster parents into the "deserving" female working and caring underclass. Relying on two years of participant observations and interviews with foster parents, experts, guardians, social workers and foster parent agency personnel, I describe the highly skilled care work most foster parents provide in demanding circumstances for a practically endless number of work hours. I explain how their status has been transformed from being volunteers to being contracted employees who work in increasingly precarious circumstances for extremely low wages. I argue that sentimentalization of care work is used by policy makers to discipline foster mothers into accepting the new terms of their relationship and by foster parents too to rationalize their compliance. Keywords Carefare • Foster care • Welfare state • Child protection • Gender inequality • Professionalization • Labor control In Chap. 2, I argued that since 2010 the Hungarian government has created a carefare regime, that is, introduced a set of policies, political practice and discourse which exacerbate the exploitation of women through their care work and in the paid labor market. In turn, the government utilizes this process to legitimate and maintain an anti-liberal rule. This chapter is about foster parents, their work, their skills, their wages and work conditions. A recent change in the regulation of fostering provides a classic example of the emergence of principles of carefare, it is thus worth studying the process in depth. Until 2014, most Hungarian foster parents worked as volunteers but were then reclassifed as gendered carefare workers with employment contracts, wages, social security benefts and increasingly diffcult work conditions. They have become part of the predominantly female "deserving" underclass-in-formation that the previous chapter foreshadowed. Hungary's carefare regime did not simply withdraw state funding from child protection. Instead the government has "creatively" (Bátory 2016) recycled it for future political gain. The meager national child protection budget has been put to use in a way that strengthens the political power of the regime and feeds its loyalists more than it feeds abandoned children. The state budget for child protection and foster care is co-utilized to reproduce political power. After a brief look at the history of child protection in Hungary this chapter describes the turn toward carefare, both within the state organization of fostering, and in the everyday world of foster parents. # Fostering in Hungary: A Quick Look to the Past Hungarian children grow up reading the story of "Árvácska" [Little Orphan] a literary classic of Zsigmond Móricz, which describes the long list of abuses suffered by a poor orphan girl at the hands of foster parents in the 1930s. Hungary's frst child protection legislation was passed in 1901, three decades before her story and the new law institutionalized state responsibilities for abandoned children (Demény 2015; Herczog 1998; Mészáros-Tóth 2014; Veczkó 2000). A central state-run orphanage was established in Budapest and the state recruited foster parents or "tápszülők", literally "feeding parents", who received payment for taking in children. Over 80% of abandoned children were raised in foster families in the frst decades of the twentieth century. Life in these foster homes, as evidenced by the story of Árvácska, was notorious for its hardships, heavy workload and vile treatment. Hungarian orphanages were also vastly underfunded, even compared to similar institutions within the Austro-Hungarian Monarchy (Varsa 2020). After World War II, state socialist policy makers reconceptualized the needs of abandoned children altogether: the communist regime wanted to put an immediate end to individual fostering and emphasized its association with child labor in politically objectionable "kulak" (rich peasant) families in the countryside. As a result and following famous Soviet author and pedagogue Makarenko's ideas described in his widely popular Pedagogical Poem, state authorities saw a chance in abandoned children to realize their dream of communal upbringing in the service of producing the new communist men (later women as well). Replacing foster care, children who were removed from their homes for a variety of reasons, including material hardship, health and moral abandonment, were sent to newly built state-run institutions (Varsa 2020). This new "scientifc pedagogical" model was understood to be the most modern way to raise children, and institutional care was considered politically more trustworthy than individual families. In addition, the supply of women at home who would have time to devote to raising children dwindled as state socialist policies pushed everyone to take on paid work. To meet children's needs so defned, state authorities nationalized several large mansions that had belonged to the upper bourgeoisie and turned them into children's homes. This move was to serve a double purpose: to strip upper class Hungarian families of their private property, and to demonstrate the regime's commitment to the most vulnerable. What could serve as better demonstration of the ideological direction of the new political regime than stories about previously starving and disheveled children playing happily in gorgeous playgrounds and parks of the kind they could never imagine even approaching before. To this day, a number of state homes for abandoned children can be found in these now decrepit villas surrounded by beautiful parks with century-old trees, some in the most sought after locations in and around Budapest. As a model solution, in 1957 Hungarian state authorities opened "Children's City" 20 kilometers north of Budapest in what used to be a castle of the Károlyi family and its surrounding 140 ha park. In its heyday the complex housed some 800 children along with 200 social workers and teachers and had its own schools, infrmary, lake and park with rare and protected trees, movie theater, sport courts and other services on its premises. Like many infrastructural establishments, children's newly appropriated homes in the early 1950s may have been considered "modern" at the time they were built or renovated for use but were henceforth vastly underfunded and gradually deteriorated. News of abuse and deprivation was silenced and critiques of the conditions in staterun children's homes only resurfaced after the 1990s (Varsa 2020). During the state socialist era, only about 20% of abandoned children grew up with foster parents (Herczog 1998), a steep decline from the 80% a few decades earlier. Children were only placed in individual homes if and when places in institutions were not available. (See state regulation 2111/1954 (VIII. 25) MT.) However, in the 1980s a slow change started in how the needs of abandoned children were understood. By this time it became obvious that the political agenda of educating children as model communist citizens had failed. Psychological research started to gain prominence in the early 1970s, and studies showed a high rate of criminal behavior and addictions among children who grew up in childcare centers (Demény 2015). The more progressive psychologists argued, based on local experience as well as increasing contact with Western European experts, that children do better if they are raised in families or at least in more intimate settings (Veczkó 2000). In addition, the mansions of the 1950s started to crumble and their maintenance proved to be an insurmountable cost. As a result, attempts started to resurrect elements of the foster care system and larger state institutions were broken down into smaller, "family like" units. The fall of the communist regime in 1989 accelerated the pursuit of these ideas already familiar to more progressive groups among child protection experts. In 1991 Hungary ratifed the UN Convention on the Rights of the Child and Hungarian experts and social work professionals looked to Western European models, primarily in Anglo-Saxon areas, to overhaul the Hungarian child protection system (author's interview with Mária Herczog, 2015). After a period of intense debate, a new Child Protection Law was passed in 1997 which set the tone for further developments. Following the lead of the UN Convention and the existing international wisdom on children's needs, this legislation strengthened the rights of birth families and established a basic framework of social support for families in need to prevent the removal of children by providing basic services once children were identifed as in danger. The new law prohibited taking children away from their birth families for fnancial reasons, and state agencies were tasked to meet the fnancial needs and basic services required by troubled families. Social services were decentralized, local providers were to offer services and local agencies determined and met needs. This legislation was one of the frst of its kind in the Central and East European region, and one most similar to existing Western principles. Then in 2014 the Orbán government passed a new law which radically transformed the Hungarian foster care system again; this will be the subject of discussion in this chapter. First, in line with EU and UN recommendations, the law stipulated that children below 12 years of age should grow up in foster (or, in ideal situations, adoptive) families rather than in state institutions. Second, the work of fostering was reclassifed from a volunteer unpaid activity into the category of a special paid employment relationship outside of the remit of the Hungarian Labor Code. A number of related regulatory mechanisms were further introduced to change the working lives of foster parents as well as of the agencies overseeing their work. There are about 20,000 children registered in child protection in Hungary in 2020 and 14,000 of them are growing up in approximately 5500 foster homes. The number of children has been increasing, while the number of foster parents remained stable in the decade of the 2010s (KSH 2019a). Child protection and within that fostering is a small segment of the state apparatus but one that vividly demonstrates the real-life functioning of a carefare regime. In the next section I describe changes in the role of the state in regulating child protection. Then in the following part of this chapter I examine how the lives of foster parents have been transformed. # Centralizing and Re-engineering State Services Austerity has been one of the guiding principles of all state institutions in the past 25 years in Hungary, child welfare being no exception. A key reason why the institutional shift in fostering was initiated and successfully pursued in 2014 was the fact that it was considered cheaper in the long run than fnancing large state-owned institutions, with their crumbling walls and decrepit furniture. In the state socialist era, the villas appropriated for children in the 1950s were barely maintained and now required major renovation. Or, alternatively, they offered an opportunity, since instead of restoration they could be sold on the prime real estate market. Soon enough, even the largest institution, the one described above in the city of Fót, was sold to cronies of the government, with children moved to smaller institutions and foster care. "The costs of care with foster parents is about 1 million HUF per year, while in a children's home, small or large, it amounts to 2.8 to 4 million HUF per year per child" (Author's interview with a high-level state executive in the Ministry of Human Resources, June 2016).1 Others, however, claimed that good quality fostering was <sup>1</sup>The quotations in this chapter are from interviews conducted during my research on fostering. See more information on the methodology in Chap. 1. just as expensive as care in state institutions, but this quality was not reached in Hungary exactly because of the scarcity of funding available. Indeed, all agencies complained about the low level of staffng, where those supporting foster parents (advisors, guardians) typically look after many more children than is mandated by the state legislation, and where funding is not available for each agency to have its own psychologist, even though all children removed from their birth homes would need one. To explain why she is about to quit her job and look for employment in a different sector, a social worker in a large foster parent network agency told me: "I know what each and every family would need to survive, I usually know it very well. But those services don't exist, so I more often than not can't help them. That's what's really frustrating" (Marina, foster parent advisor, Budapest). How much did the state spend on child protection after the transformations in 2014? That is diffcult to tell. Unlike neoliberal effciencyoriented work organizations with their audits, benchmarks and indicators (Shore 2008), transparent data collection and presentation is not of high importance for anti-liberal rulers. In principle, state spending on child protection is public information in Hungary. But getting reliable and systematic data requires a very long wait, personal connections and favors. I managed to obtain some, but only some of the required information from the Hungarian Treasury for the years of 2010–2017 and a selection of the relevant line items is included in Table 3.1. The data are diffcult to interpret because, as experts at the Treasury warned me, the rules and principles of data collection regarding child protection services changed twice during this seven-year period. In 2013, the data collection on government spending was revamped and activities were reclassifed in a way that made comparison with later years impossible (hence the gray shades in Table 3.1). In 2014 the child protection system was completely transformed based partly on the argument that foster care was cheaper than institutional care. Yet at least for the years 2014 and 2015 it is impossible to separate the amounts spent on fostering and on institutional care—they are grouped together as per the regulation of 2013. The rules changed again in 2016 and spending data are again available in detail. Whether or not the categories cover the same expenses is altogether unclear and information on this was simply not accessible. The meaning of a category "Programs supporting the life quality of children and youth", which had no allocation in 2016 but amounts to over 10% of Note: \Includes both institutional and foster care Source: National budget accounts (Hungarian State Treasury) the overall child protection budget in 2017, is impossible to penetrate. And the classifcation rules changed again in 2020. With these caveats in mind, what do we learn about possible changes in the amount of state spending on child protection over the years? Shall we describe this as a period of grave austerity? To the extent that this is discernible, given the lack of transparency in the data collection and presentation, there is no sign of state retrenchment for the child protection sector as a whole. Instead, we see fuctuation—possibly due to classifcation changes or to actual cost-cutting—and some overall growth toward the end of the period. Between 2010 and 2013 the data show stagnation and decline in spending for the frst three years then an 8% growth in the fourth, in 2013. The overall four-year change is smaller than the cumulative infation rate over this period, which means that the real value of spending stagnated at best, most likely declined. After the frst major change in the classifcation system in 2014 note the sizeable fuctuation over the years and a sudden increase in 2017. Some of this increase has to do with the rising cost of nursery schools, which is also included in this rubric, but there seems to be a general tendency toward increased spending on vulnerable children in every category of the table. There is one exception as the third line in the table attests: foster care. While the number of children in foster care increased by over 25% between 2010 and 2017, spending on fostering recorded in the central budget remains unchanged. This, given the rate of infation, means a signifcant, roughly 30% decline in real value. However, there is another budget line, "state support to non-state providers", which is the source of additional funding for some foster care agencies. I explain these below. Contrary to the deregulation tendencies of neoliberal states, the Hungarian government has centralized the management and fnancial control of child protection: in 2012 it wrestled responsibilities and power away from county seats and local governments and established a new institution of the Ministry of Human Resources, which is tasked with the oversight of child and family protection services. Yet simultaneously, another type of deregulation was initiated. Neoliberal states encourage commodifcation with the assumption that market-based distribution and provisions are the most effcient, or at least the cheapest (Wacquant 2012). In a process counter to this logic, the Hungarian state has "churchifed" child protection: it allocated signifcant resources toward the establishment and fnancing of Church organizations based providers. The penultimate line in Table 3.1 demonstrates that funding to such providers almost quadrupled over the seven-year period and has increased further since. The impact is clearly noticeable too: in 2010 about 7% of children grew up in church-affliated foster parent networks, but by 2018, almost half, 47% of them did (KSH 2019b). Practically all large established churches operate foster parent agencies in 2020: I counted 17 different church-based and 3 civil or international networks in addition to the state's agency. It is not merely out of a calling to help the down-trodden that churches have so successfully got involved in the business of child protection. Hungary's anti-liberal government pays a quota for each child in the child protection system to the state provider, yet pays an additional 70% of this amount as extra for each child who belongs to a church-affliated agency. This is the sum in the line "state support for non-state providers" in Table 3.1. Ninety-seven percent of the money allocated in this rubric goes to religious organizations. The basic head quota must be allocated toward the designated service, in this case, child protection or fostering, even if it is managed by a church-affliated provider. But churches do not have to give account of how they spend the additional funding: it may or may not go toward the care of children. Foster parent network directors, whose organizations came to be affliated with churches as a survival strategy after this legislative change, told me that their organization received between 10% and 20% less than the full state allocation—this is the amount that the church keeps for its own budget of what is technically allocated for "child protection services". In addition, a legislation was passed in early 2021, which gives church organizations running social services, including child protection, property rights over the real estate in which they are currently operating. In the frst round in 2021, 29 real estate properties were passed on to various churches by the Hungarian state. This also means that the churches are now eligible to apply for and receive funding from the European Union to renovate the buildings, some of which have exceptional value. This increases the wealth and political role of churches that are hegemonically loyal to the government. It also allows the state to channel EU funding to organizations over which it has signifcant control. As I noted above church organizations use most but not all of the allocated state funding to support their foster parent networks. Their insistence on the religious education of children varies from a tolerance of positions to a clearly stated expectation of participation in religious services. My feld notes from a conversation with social workers in a recently church-affliated agency describe the position of the agency's leader: We switched affliations in 2012 and since then [the Church] is our maintainer. Every week a representative from the Church visits to discuss everyday issues. Practicing the religion is not compulsory but they did insist that children follow their religious practices in the summer camps. At the same time, they are not forcing us to change our professional work because of their faith. (Director of foster parent network, Budapest, Nov 3, 2016) Other churches are signifcantly more demanding. I talked to the extremely professional and compassionate manager of a then relatively small foster parent agency run by the Catholic Church. She was quite insistent on practicing the faith because she considered it a better way of living. It is not compulsory for foster parents to be familiar with the teachings of the Catholic Church. We cannot hold them responsible for that … but we would like them to be aware. I mean aware of Jesus' mission, the basic values of the Church, we teach those. This is not proselytizing … we obviously don't do that. But there is a softer version of evangelization, for example, that we celebrate religious holidays and include the children and the foster parents and we celebrate together. Or we have these obligatory foster parent trainings. The next one will be taught by [a well known Catholic priest], so there is certainly an infuence, not forceful, but it is important to pass on our values. (Head of foster parent agency, Budapest, 2016) The Protestant Church's foster parent network is based not only on faith but requires belonging to, or at least being familiar with, local church organizations. The head of the agency explained: You can only become a foster parent if you have a recommendation letter from your local minister. This is a guarantee that the person is not doing it for the money. And it is important for the children to join the church community, because those who belong there are more likely to lead a Christian life, a solid, stable, organized, harmonious life. (Head of foster parent agency, 2017) The principle of the separation of the state and church is clearly not of high importance here. The political gains are obvious. First, FIDESZ governs in coalition with the remnants of the Hungarian Christian Democratic Party, and even though the latter has little independence in most matters political, they tend to contribute to the discourse on family, children, gender, morality and so on. Also, surveys have shown that the governing parties are signifcantly more popular among those who claim that they are religious, so measures promoting religious organizations is a form of catering to the demands of the electorate, indeed, creating these demands in the hope of reaping electoral benefts later (Policy Solutions 2015). In addition, media accounts describe numerous instances when "recommendations" on voting were shared from pulpits before elections—making the support of churches an eminently sensible political investment. In summary, it is diffcult to interpret data on the volume of state funding for child protection. It is clear that we cannot talk about major cuts in funding, but neither is there any sign of additional support from a supposedly family friendly state to a growing number of needy children and those who take care of them. What is absolutely clear, however, is that in recent years a signifcant chunk of child protection services along with their funding have been outsourced to loyal church organizations. The everyday control of these institutions is variable but the potential for comprehensive oversight is there. The churches receive additional funding and bonus real estate from the state to encourage their participation. Then they can use the opportunity to expand their networks, their follower base and evangelize, if desired. The state, on the other hand, spends additional taxpayer money on offering religion-bound services and buying political loyalty from and via church organizations. # Carefare: The Work of Fostering Along with this faith-oriented re-engineering of state functions, the position of foster parents vis-à vis said state has also changed. Before 2014 with the exception of a handful who qualifed as paid social workers, the vast majority of foster parents worked as volunteers and provided a service to the state out of kindness and dedication. This changed abruptly when a new piece of legislation eliminated the volunteer foster parent category and transformed foster parent contracts into paid employment relationships. As of January 2014, foster parents have become "professionals". They are working for wages at a designated foster parent agency, receive a set salary and occasional bonuses as well as social security coverage, including old age pension benefts. This may sound like a turn for the better for the kind volunteers. But below I argue that using the pretext of "professionalization", foster parents have in fact been hired to work on the principle of carefare: they have become part of the working female underclass. Relying on two years of participant observations and about 80 interviews with foster parents, experts, guardians, social workers and foster parent agency personnel, this chapter will demonstrate that foster parents provide highly skilled care work in extremely demanding circumstances for a practically endless number of work hours, often for wages amounting to less than the national minimum. Having been classifed as "workers" means an increase in control and surveillance on the part of their employers, less independence and more supervision for the foster parents themselves. This process of integration into carefare was described as "professionalization". ### Professionalization* In the Parliamentary debate on the legislative changes involving the status of foster parents, Mrs. Ronaszeki, who introduced the bill and was an MP for the government and member of the Committee on Youth, Social, Family, and Housing Affairs, pointed out that "It [was] important for foster parents to turn their relationship to the state into one of employment in order to 'professionalize' the activity and increase its social prestige" (Mrs Ronaszeki, 2013 in the Hungarian Parliament). She was not alone. Child protection experts had long argued that the social context of fostering had changed and required more skills, expertise and energy on the part of the carers. The children are more vulnerable, it used to be much easier. We don't do well at the early stages of the [child protection] process, when they are registered in the system, there's not enough help. So the children arrive in the system really worn out … even 3–4 year old kids need therapy. (Foster parent advisor at a Budapest agency) In sociological parlance, professionalization is the process of creating distinctions amongst those who belong to a specifc occupational group and those who do not, between "professionals" and "amateurs". Since the Middle Ages occupational groups have been fghting to establish themselves as professions, a position, which typically brings distinct privileges, such as higher earnings, the possibility to claim monopoly over access to clients, as well as respectability and status (MacDonald 1995; Wilensky 1964). The establishment of a profession is often a contentious process as was, for example, the case for midwives (Bourgeault 2006), or librarians (Abbott 1988). What makes a professional is widely contested: some acknowledge their expert knowledge, established practice of the trade and self-regulating professional associations (Parsons 1968), while others refer to their position in the social hierarchy which allows them to exclude others and construct themselves as members of an exclusive, elite group (Abbott 1988; Larson 2012). An altogether different process of professionalization has taken place in the case of Hungarian foster parents: it has been initiated and enforced by the state. Foster parents did not claim to be professionals, quite the opposite, many of them actively resisted the term. Yet state policies, new legal regulations and institutions have decided to construct them as such, and they have allocated some dubious distinctions while simultaneously imposing a new set of expectations and obligations. Historically, there have been other instances of professionalization that involved more than just grassroots actors. McClelland (1991), for example, describes "professionalization from above" in nineteenth-century Germany, where the state had an important role in the regulation of entry into professions, such as medicine and law, even engineering and chemistry, as distinct from what he calls the more autonomous "professionalization from within" process of the Anglo-Saxon model. Our current case of the professionalization of the child protection system in Hungary is an extreme version of professionalization from above, where those proclaimed to be professionals had little input into a process shaped instead by politicians, policy makers, as well as local and international experts. Professionalization targets foster parents who are expected to transform themselves from warm-hearted women raising children in need, to professional paid carers with expertise and lengthy training. They must adjust their work schedules to satisfy these criteria, enroll in specialized training programs and write lengthy dissertations and reconfgure the way they raise children to ft the principles of childcare considered suitable for the "modern" world by experts on child development. They must also meet new institutional expectations as professional carers and subject themselves to even more supervision and surveillance than before, while simultaneously losing further degrees of control over their work process to the requirements of professionalized processes. In administrative terms, foster parents must enter a formal employment relationship with a network agency; for better or worse they become part of the formal labor market. In fact, professionalization from above can be understood as a new form of labor control exercised by various state authorities over foster parents who are employees working in a context where typical methods of supervision are not easily applicable. Researchers have described different mechanisms of increased supervision and coercion, such as scripting or digital automation (Wharton et al. 2008), emerging in the post-Fordist economy. But there are limits to the possibility of despotic control in a setting where clients and customers also feature in and complicate the labor process (Leidner 1993; Sallaz 2015; Sherman 2007). In such contexts other mechanisms such as "permanent pedagogy" (Sallaz 2015) or "relational work" (Mears 2015) function as substitutes. How to regulate the work of people who do it in their own homes, however? Fournier (1999: 281) argues that fexible work practices create a "discretionary gap" which "needs to be regulated through new softwares of control. Professionalism is one of the strategies deployed to control the increasing margin of indeterminacy or fexibility in work." Professionalization from above is thus not a politically innocent strategy. "It is through their 'professionalization', through their inscription into systems of expert knowledge, that individuals become targets of liberal government" (Fournier 1999: 284; also Burchell et al. 2014) as well as targets of anti-liberal government, we must add. Professionalization from above, I argue, is the way in which foster parents get more tightly integrated into the lowest rungs of the carefare regime, often against their explicit will, occasionally with their informed, or uninformed consent. ### Beyond Parenting It is easy to assume that fostering is nothing but the kind of regular parenting that millions of people do, most of them in addition to their paid jobs. But a closer look at the daily activities of an even mildly conscientious foster parent quickly proves this statement wrong. As one foster parent, a mother of three biological children, succinctly put it: "You'd think you know what you're doing until you get a foster child. Then really basic issues come up that you had never encountered before, stuff you'd never even dreamt of" (Ibolya, birth parent of three, adoptive parent of one, foster parent of two). Foster parenting is extremely taxing work. For one, it requires being on call 24 hours of the day on every day of the week and it opens up one's home to ongoing scrutiny from others. More importantly, fostering requires a number of special skills. Below I describe three sets of these relying on the accounts of the practitioners themselves: (1) a working understanding of child psychology and the management of emotions—their own and those of others, (2) advocacy skills to be exercised in adverse conditions, and (3) an exceptional level of understanding and daily practice of logistics and administration. We all need and rely on these skills in our everyday lives. But the stories below demonstrate Ibolya's claim: none of us are expected to use these with the intensity and within the specifc conditions that foster parents do. #### Child Psychology and Emotional Work Children arrive in foster homes from a variety of domestic or institutional circumstances and in varied mental and physical conditions, and the encounter is rarely simple. The child is typically traumatized not only from her past experiences but also from having been removed from her previous surroundings, landing in a rather different setting often all alone, and having to adjust to yet another set of rules and constraints. The foster parent and her family, much as they may be expecting the child, have to make adjustments and many recognize the process as diffcult. The frst task of the foster parent is to help the child adjust to his or her new circumstances, which in the case of deeply traumatized children is not a simple task. Edina, a foster parent with a great deal of practice described the arrival of her three-year-old daughter: She had a rather hectic background at birth, poor baby, and we were up all night for, I am not exaggerating, at least 6 months. She screamed through the night, no matter what we did. It was horrible. That was the worst. (Edina, three foster children) Foster Parent Network Agencies do not exactly expect parents to treat children with psychological needs, "merely" to handle them with tact, kindness and understanding. They should be able to recognize problems, seek help and then follow the treatment suggested by the experts. But the hands-on support the agencies can afford to provide is rather limited. Even the largest foster parent agencies have a single in-house psychologist in their employment and some do not have any at all. Even when there is a psychologist working for the agency, they have a variety of tasks related to institutional needs. They must, for example, participate in the evaluation and screening of foster parent applicants. Their time to work with children and their carers is thus limited. One psychologist who had spent several years at one of the large agencies told me that her job required total devotion, daily traveling of several hundred kilometers as well as endless work hours. "You can only do this if you are a saint and I am no saint", she said explaining why she quit the job she loved after a few years. Given the limitation in resources available through formal channels, foster parents often use their own practical experience and knowhow to deal with at least the easier problems, and these are numerous. The example below comes from a very experienced, loving foster parent: They [her two fostered children, who are siblings] are different in many ways. … But they both come with a big baggage. Viki, for example, has a number of unexplainable fears of things. And bedwetting. Tomi was six years old when he got out of diapers and Viki is well past six yet she wets her bed nightly so I put a diaper on her to sleep. (Eszter, Pest county, foster parent of two) I heard countless accounts of acts of destruction from spreading feces on the wall with regularity to throwing heavy objects at television sets. The foster parent is expected to calm the child, quietly clean up and try to deal with the cause of the problem. No damages can be claimed and this behavior is not considered out of the ordinary or as needing external support. Older children express their anxieties in different ways and foster parents must work not only with their own families but also with their broader environment to make adjustments. This can be especially diffcult in smaller settlements where families know each other. This was the case of one foster parent who lives in a village some 60 kilometers east of Budapest when his foster children, a pair of siblings, arrived about fve years ago. He is still embarrassed about being the "talk of the town" even though the problem was in fact resolved. At the beginning Dani [his fostered son] stole other kids' snacks and this came up at a meeting of parents at school. I told them [about the background of the child]. So the parents then understood and I am sure, well, I hope that they explained it to their own children and then this stopped. (Gabor, fostering two school age children and parenting biological twins who attend the same school a few years ahead of the fostered siblings) This foster parent had to fnd the right way to discuss the issue within the community, make sure the child and his own family are not excluded from the village for their unruly behavior and negotiate the child's needs which resulted in his stealing snacks. He only had perfunctory help from the foster parent agency to tackle the problem and mostly had to rely on his own skills as a negotiator, mediator and child psychologist. Another foster parent describes the way she "tamed" her son, Alex, who came with serious mental and psychological diffculties. I have managed to get him to the point where he doesn't bite or kick, where tools and other objects are not fying around … so you see, this is a frst step. Now he only screams. Which is better than when he threw half a brick at me. (Nikolett, foster mom to 2 children, with a great deal of fostering experience) Alex's rage was successfully controlled by this foster parent without the help of an expert psychologist. She relied on her research online, the advice of her social worker, as well as her experience with previous children, to get to the point where the child was no longer destructive, a feat previous carers in institutions or private homes had not been able to accomplish. Some people foster children with known disabilities, for example, Edit, who decided to do this work for the purpose of helping sick children. Both of her fostered toddlers have Down syndrome yet she received no training in how to raise children with Down. I looked it up at the Down Foundation in Budapest. The frst step was that we went to a clinic for children with Down syndrome. That's in Budapest. There they told me in detail what medical tests need to be done. No one had explained that to me within the [foster parent network] agency. If I had had to wait for them, we still wouldn't have gotten anything done. Because I think they themselves don't know what this [raising children with severe Down syndrome] really means. (Edit, fostering two children, in a small town in Pest county, about 80 kilometers from Budapest) Her case may be one of the extremes, but for all, ongoing attention, tolerance and intense emotional and physical work are required. A small fraction of foster parents can afford to pay for psychologists out of their own pockets, others may lobby networks to help them cover the extra costs. Many said that they read books on childrearing and popular psychology, and browse internet websites in search of possible solutions to behavioral/psychological problems. They may also consult with the social worker in their children's school as well as with the agency's foster parent advisor, who visits regularly and advises foster parents on all issues related to caring for their wards. They participate in supervision and small group training workshops in at least some of the foster network agencies and discuss problems with other foster parents. Altogether, foster parents accrue a great deal of practical knowledge in child development and psychology and are expected to utilize these skills in their everyday work of raising children, who typically have grave social and psychological disadvantages which must be tolerated, handled and in the best scenario, treated successfully. Never is the need for skillful emotional work more acute or evident than before, during and after the regular visitation meetings between birth parents and their children. These visits are required by law and happen at regular intervals, typically bi-monthly, or monthly as per the agreement between the courts and the birth family. In Hungary the management of these visits is the responsibility of the foster parent, although about half of the time the actual encounter happens in a location designated by the agency with some professional personnel and supervision present. Some birth parents only show up irregularly and sometimes skip visitations without advance notice. This makes for wasted trips and even worse, bitterly disappointed children. One foster parent talked about how he took his children outside so they can yell their feelings into the air—a technique she gleaned from a book on child psychology. Foster parents often develop elaborate strategies to avoid these disappointments, from not telling the child in advance about the meeting, to planning fun events to be done near the meeting place in case the birth parents do not show up. One time we set off with the two kids [for Budapest to meet birth parents] and when we were [entering Budapest] they called to say they couldn't make it because they got sick. So imagine this child, who had carefully prepared a drawing [for her birth mom], who hadn't seen her parents in 6 weeks or so and then we fnally leave and we are almost in Budapest. At every village she asked "is this Budapest already?" And then they tell us not to bother going. We turned back and we simply couldn't comfort Barbie, the older child. It was really hard on her. That and the next few days, those are always hard. The kids somehow can sense it that the four weeks are up, even though we try not to talk about [the upcoming meeting with the birth parents], just in case [it gets cancelled].They can sense that it's time for the meeting and they behave accordingly. (Gabor, foster parent of two, who takes the children for visits once a month and lives in a small village about 40 kilometers from Budapest) When the meeting does happen, the adjustment back to the reality of life in the foster home must be managed carefully. Birth parents may, sometimes in the best of faith, sometimes out of negligence, make promises they cannot keep, and this leads to confusion, anger and anxiety that foster parents must somehow manage. You see, the birth father takes out this child. When they meet, he promises the skies to her and then he disappears for two months. So we try to digest this. Of course then I know why the kid is going crazy, why she is throwing things around or why she bites her classmates so badly that they bleed. (Kata, foster mother to four, Budapest) Other times children may have memories of trauma in their birth homes and fnd the meetings stressful. The quote below describes this, probably coupled with the foster parent's animosity toward and fear of the birth parents because of their alcoholism, unruly behavior and anger toward her and the Foster Parent Agency. Even if the latter is taken into account it is a good example of the psychological stress produced by and to be managed at meetings (or missed meetings) with birth parents. Says Viktoria, foster mother of a baby: This child, Csilla, whom I fostered, she was terrifed of her parents. Imagine an eight-month-old child desperately hanging on to my clothes when she saw her parents. It took 2–3 days after each visit for her to calm down. When the parents were forbidden to see her for a few months she became so happy and relaxed just because she didn't have to meet with them. And no matter what I tell a psychologist, they can't do anything about it. There is a serious problem here with the legal regulation. (Viktoria, currently fostering one child) The legislation she refers to is the Child Protection Law which gives birth parents extensive rights over their birth children, which foster parents often see as unwarranted and undeserved. Indeed, most birth parents are not especially well equipped to spend two meaningful hours with their children whom they had not seen for at least two weeks. The situation in which the meeting takes place does not help matters either. The visitations are typically arranged in a large room of the foster network agency, where several other foster-birth family couples are also present. Sometimes the encounter is monitored by the foster parent herself and one or more supervisors of the foster agency. These are tumultuous affairs. I spent several months helping out at visitation hours at two different Foster Parent Network Agencies, observing and occasionally helping with these rather awkward get-togethers. Parents arrived, unpacked soft drinks and sweets, had snacks with the children but then had trouble expressing their love and devotion to their child in this heavily supervised context and in the way it was expected of them. The meeting rooms are packed with toys, so children could run amok, but birth parents rarely had the skills, the patience or the mood to play along or to simply engage with children, apart from watching and embracing them and feeding them snacks. There is no parent–child relationship between them. She'd say, come here and then embraces him but that's all, nothing more. She hands him her phone and tells him to go ahead and play. So these visits are not exactly meaningful in that way. (Mrs Csicso, long-time foster parent, currently of three children) Truly excellent foster parents take it upon themselves to manage the situation in a way that it becomes comfortable for everyone. This is diffcult because birth parents' needs must be taken into account as well, as well as the limitations in their ability or desire to parent. Here is how one foster parent of many years described what her role is during the visitations. When Moni and her parents don't know what to do with each other I try to ease the situation because it's so embarrassing to just sit there and look at each other. And of course Moni would come to me as she does on every other day of the week. And then I would tell her, come on, let's show Mummy what has happened. And I tell the mother what happened to Moni that week but I try to get her involved and get her to say something and Laci [her younger child] too, so he would say something as well about what happened. And I ask the parents about their lives because of course they also have lots of problems. (Erika, foster mom of two in Budapest) This is the exception, rather than the rule, as cultural, class and ethnic differences—not to mention the built-in animosity on the part of the birth parent toward the Agency and its representatives for interfering in their lives—often make even simple communication between birth and foster parents diffcult. Many foster parents had no patience for dealing with birth parents whom they often blamed—directly or indirectly—for the problems the children faced. But the most successful ones took on the task of bridging the class/ethnic gap and specifcally "teaching" birth parents how to parent. This required a great deal of personal dedication and skill. Szandi's mom comes the second Saturday of every month and then we try to do something together. Because she [the mother] is a pretty neglected person, we take her to the Zoo as well, or to swim in lake Velence or Balaton. We kind of adopted her as part of the family and on that day she is also our child. So we go together. (Andrea, foster mother of two children) #### Exercising Professional Technologies of Self Professional foster parents are expected to relate to their children in a professional manner and exercise what is called "smart love" in their work activities. Broadly speaking this means loving the child without a sense of ownership and full commitment. Foster parents are taught to love their children but love them with reservations; to handle them as members of the family yet view them as temporary additions, and as people whose ultimate fate is not in their hands. Most experts agree that this is a tall order: "Foster parents must have a split personality: they are expected to tell the children that they belong there, they are members of their family, yet must also encourage them to return to their own birth parents" (Foster parent advisor, explaining why he could never do this job). Indeed, this is a diffcult balance and most foster parents are somewhat unsure about what "smart love" means or whether or not they should strive to provide it. The best explanation I heard highlights the vast amount of emotional work that goes into "smart love", well beyond a simple love for children. Well, you have to do everything the same as with your own child. That kid needs lots of love in their frst three years, so they can be self-confdent later. You must love them the same, only you have to be strong when it is time to let them go. Because it is a lot worse for them if they see that I am reluctant to let them go, then he will feel guilty. (Bori, an experienced foster parent, who has raised a number of very small children who were later adopted, as well as a few who grew up in her household.) Indeed, "losing" a child to adoption or return to their birth families is often rather traumatic to foster parents many of whom grow to genuinely love their children. This requires so much emotional work that some agencies offer psychological counseling on the otherwise rare occasion when a child leaves. Knowledge of child psychology, complicated emotional work and tolerance of psychological distress are all practiced by good foster parents on a daily basis. While the work itself is often acknowledged by agency personnel, the skills that go into managing the foster child–foster parent–birth parent triad is rarely noted (for an exception see Demény 2015). #### Advocacy Within Boundaries While foster agencies do not expect foster parents to be able to solve all the child's psychological and learning problems, they do expect them to advocate for the child in various contexts. This is harder than expected because foster children are often surrounded by discrimination and distrust, both because of their status as protected children and because many of them belong to Hungary's largest minority, the Roma. Hungarians in general express a great deal of animosity toward minorities of all stripes. Anti-Roma sentiments are especially strong and have increased over the past decade. In recent surveys 73% of the population said that they would not consent to a member of the Roma minority moving into their neighborhood, and news and academic reports describe increasing violence against the Roma (FXB 2014). Terms, such as "Roma criminality" abound in the media and the supposedly problematic "lifestyle" of Roma groups is routinely pathologized by Hungarian politicians. In this context, advocating or simply standing up for the rights of Roma foster children requires exceptional courage and determination. Advocacy is especially hard as foster parents have limited rights over the child: the fnal decision maker is the child's guardian. In addition, foster mothers—who have typically graduated from a technical high school with a certifcate in a specifc trade—have signifcantly less cultural capital than the teachers and doctors they must negotiate with. It is no surprise then that the ability to advocate for their child was one of the key requirements listed by agencies when recruiting foster parents. In fact, this is one task agencies typically claim is "work"-like. As Sára, a foster parent advisor told me: For this [foster] child to be able to persuade people that she is valuable, not a waste, she must be at least beautiful. But if she is naughty, and ugly and god forbid, Roma and maybe even steals occasionally, poor thing, then the parents in the school will collect signatures against her … so the [foster parent] will have to lobby hard for her … she will have to represent the child's interests. (Sára, foster parent advisor, large network in Budapest) At the same time foster parents must act with a great degree of decorum and be careful to behave in a manner considered "civilized" by the mostly middle-class and middle-aged experts of the foster agency. They are expected to represent the child, but true "tiger moms" are frowned upon too. Ildiko, a middle-aged seamstress in a rural town fostering a small boy who was mistreated in kindergarten was told off by her advisor when she made a scene at the childcare center. Then [the foster parent advisor] told me on the phone that I was too loud and I didn't behave appropriately and this behavior is not suitable for a foster parent. So I said, yes? I would have been curious to see what you would have done if they had treated your child like this, what would you have done? Of course foster parents must stand up for their children, how could I not? Stop kidding me, should I just laugh when they mistreat him? Come on … I said that was out of the question, don't even say such things to me. (Ildiko, foster mom of one, her two biological children have already left home) Worse than frowned upon, in fact, this woman was threatened with the removal of her child when she raised hell for what she perceived as unfair treatment bordering on violence. Foster parents are expected to represent the child's interest vehemently, but within what is a typically moving target of "professional boundaries", something that is not necessarily part of the vocabulary of lower middle-class blue—or white-collar families. Here's another example from a woman who talks about the same problem though formulated in different terms: You as a foster parent cannot act as a "tiger mom". Because in the end I have no rights at all, all I can do is shut up and raise the child. If I don't do something perfectly, the child may get taken away, in fact even my own kids may be taken. (Viktoria, foster mother of infants, birth mother of two, living in a rural town) The foster parent above may be expressing more anxiety than is probably warranted, but her point is on target: foster parents must navigate between the Scylla of middle-class civility and the Charybdis of vehement advocacy in diffcult situations. This requires an understanding of how institutions work, refned interpersonal skills, self-control and perseverance. Dealing with authorities when children have health problems or run-ins with the police are similarly diffcult. Says Erika, who raises six children in a small rural town and teaches religion in kindergartens part-time: [There was a period of time in the life of her fostered daughter when] I spent more of the nights at the police station than in my own bed. But what really broke the camel's back was when she started using drugs. So I told her to stop, everyone else did too, the whole foster parent network came to talk to her. Erika was barely equipped to deal with police issues, not to mention problems related to drugs and alcohol use. She had received no training which may have taught her how to manage these issues. Neither did she get suffcient help from her advisor and agency, even though she had alerted them to the problem. They came to talk to the child, but in the end it was always Erika who had to bail her out from diffcult situations. Anita fosters a girl who just turned six when I talked to her, but she had started noticing problems when she had enrolled her in kindergarten there years earlier. She spent about 20 minutes of the interview describing her trials and tribulations during the process of getting the child some help, of which this is a short excerpt: So I took her to lots of doctors. I told this local doctor that something was wrong. He saw that too so sent us to all sorts of places, from the Child Development Service to speech therapy, I took her everywhere. [She needed permission for each expert visit, as well as fnancial support from the agency. Neither of these could be attained without lengthy petitions, numerous phone calls, explanations and occasional surprise visits to the relevant offcial's offce.] In the end, she received some developmental training and speech therapy, we did this for 2–3 years … we went to a whole list of therapy classes. (Anita, foster mother of 2) While birth parents often have to go through the same process, the incidence of developmental lags is much higher in the case of fostered children. In addition, foster parents must ask for permission from the child's guardian and the foster parent agency for every move they make, they must rely on guardians to manage the paperwork, which often takes months, and they face discrimination at every step of the way from authorities, including schools and health care providers. Foster parents must thus advocate for children in a society where discrimination against the Roma and indeed against children growing up in untraditional families of any sort are rampant and where their own relationship to the child must be negotiated among the different actors, including the child, in an ongoing manner. This is a skill acquired through the process of fostering and is developed in everyday practice. Foster parents themselves talk about learning the ropes through their own mistakes and doing better on subsequent occasions. Yet, neither the skills, the effort nor the hours are acknowledged when fostering is categorized as an unskilled job and when its wages are set. #### Managing a Foster Family Managing a family requires a great deal of invisible work: women's mental load has been described at length in both academic literature and the media (Daminger 2019). However, managing a foster family is an enterprise on an altogether different scale. Let us review some of the administrative and management duties unfamiliar to most of us. In addition to their own wages, foster parents receive an allowance to cover the living expenses of their child. The full sum must be devoted to the needs of the child and, in many although not in all agencies, the money must be placed in a separate account. Whether or not foster parents must collect receipts of everyday or only larger purchases for their wards (such as clothes or books or food) varies. In some agencies, this is the norm, and receipts are checked randomly. At other agencies, only certain items must be accounted for. Oh and the clothes money. We have a certain sum we must spend on clothes each year. We are very lucky in this regard because we don't need a separate bank account and simple receipts will do. So I have a separate folder where I keep the receipts [for each child] from clothing purchases. (Erzsébet, currently fostering four children) All foster children receive pocket money, which needs to be accounted for. In fact, each child starting at the age of three must sign a form each month to acknowledge that they have been given this amount. The foster parent advisor, during her bi-weekly or monthly visit, regularly asks children what they do with their pocket money as a way to check on whether or not they have received it. Foster parents, as a result, develop a variety of techniques to make sure the child understands that his or her money is spent on the toy or food item he chooses and they try to imprint this on the child's memory. During my interviews at a foster parent agency, I was shown the dossier of a foster parent, which contained a long list of days with signatures. This was required because two of the children raised in this family studied at a live-in high school in a different town. This meant that the foster parents only got a part of their salary, proportionate to the actual time the child spent in their homes rather than in the dorm. These days had to be documented on a separate sheet, every month, and fled with the foster agency. Finally, foster parents must keep a diary of the life of their foster child in view of the fact that they may be able to go back to their birth homes or may get adopted. Here's the diary, let me show you. It is about the children, exactly because they are here temporarily—so to say. If they move on to anywhere, are adopted or able to move back to their birth parents, I must be able to show what happened to the children while they lived with me, how did he get to where he is now. This is his life … it is part of our contract, one of our tasks, to have this life history diary. We must do it. Now, we can decide how we do it. This one (she is showing me the book) I wrote in this every day at frst, later only once a month. And then he made drawings. This was written to them by their parents, I insert these as well, all sorts of experiences. Now I am starting to add photographs as well. I just thought it's much more practical from my point of view to write down if something happened immediately that evening, rather than go back to it in a month. (Ilona, foster mother of three) Not all foster parents are as conscientious as Ilona, and some simply keep pictures of the child's life in a folder on a computer or smart phone. I've seen many variations of life diaries as foster parents are typically proud of their children and celebrate their achievements with enthusiasm. The documentation varies in depth and quantity and it must be made available at the request of the foster parent advisor. This is in addition to the biannual reports that are fled by the child's guardian, but are in fact typically compiled at least in part by the foster parent herself. While birth parents may be somewhat careless with the personal documents of their children, foster parents must have all personal and legal documents as well as the history of the child's life organized and potentially accessible at any time. Administrative work is a hidden aspect of all parenting, but the lives of fostered children need extensive and in-depth documentation, which must be made available for scrutiny at a moment's notice. Indeed, foster parents are now trained in the legal and administrative aspects of their jobs in the preparatory fostering courses they are required to take. The newly introduced employment contract brings further administrative burdens for the parent as an employee, and this requires regular revisions, because wages fuctuate with the number of children in the home and other minor changes. In summary, fostering is extremely diffcult work, which requires the kinds of skills typically associated with women and especially mothers: emotional work, caring and advocating for others, negotiating, administering, organizing and managing people's lives. As women's work it is often seen as being a "natural skill". But as the foster parents themselves have described above, the contexts in which they do their work, and their overall work burden are hardly simple extensions of women's feminine selves: they are examples of highly skilled labor, exhausting the body and the soul. It is thus especially appalling to understand the wages and work conditions of foster parents, which I argue place them squarely into the category of the carefare underclass. #### Wages Over 90% of active foster parents are women, and while several men are also certifed as part of a foster family, only exceptionally can men without female spouses house fostered children. Single men in fact face a great deal of suspicion and discrimination when they aspire to the job, and foster parent advisors have asserted several times that fostering is really for women. This is relevant because female-typed care occupations (such as teaching, childcare or nursing) are especially devalued in Hungary, as indeed they are internationally (England 2005). As one foster parent said and this was not at all meant as a joke: "This is women's work, because men can't bear to work this much". Formal educational requirements are fairly low. Foster parents are expected to have completed elementary education only, but then they have to take a special skills training course of 500–600 hours and pass an examination at the end. Experience in successful childrearing is important in the selection process. Some agencies explicitly require that applicants will have raised a child of their own. They consider this important to demonstrate that the foster parent applicants are aware of the job of parenting, they have a track record of having raised children and also so they are less likely to think of foster children as their own. While everyone involved agrees that fostering is extremely diffcult and complex work, most foster parents earn less than the national minimum wage. Their salaries are set by state regulations and foster parents working for all agencies and with different levels of experience get the same amount, although some agencies may be more or less generous with covering special costs for children or distributing an occasional bonus payment. All foster parents earn 30% of the national minimum wage, and another 20% of the national minimum is added for each child they foster. This means that a foster parent makes only 90% of the national minimum wage if they foster three children, which is the typical number in Hungary.2 In addition, women who foster children under age 2 may also receive parental leave benefts on top of their wages. Interestingly, while this beneft is set at 70% of the minimum wage for other women who do not have a higher income of their own (e.g., university students), foster parents only receive 50%. Note that there are two types of minimum wage settings in Hungary: one for everyone, and another for skilled workers whose skill is required on the job. Many foster parents have secondary school qualifcations and in any case they are expected to graduate from a year-long training course which endows them with a diverse number of specialized skills—as the syllabus of the program attests. As we have seen above, they employ all those skills and more in their everyday work. Yet the minimum wage that applies to them is the generic national minimum, not the specialized one, which would be 30% higher. Importantly, no distinction is made among foster parents by their educational level or experience, as is customary in other segments of the labor market. The only addition is an extra 5% of the minimum wage if a foster parent raises a child with special needs. This amounts to about 8000 HUF per month or 22 EUR, which is roughly the price of ten Big Macs, to use this index of purchasing power parity. <sup>2</sup>The minimum wage in Hungary is the second lowest among EU members states after Bulgaria. A foster parent who raises three children gets paid 144,900 HUF (gross, taxes are payable), which is 400 EUR per month in wages in 2020. The handful of professional foster parents already fostering before 2014 in Hungary earned more than this amount, regardless of the number of children who were placed in their households. However, fewer than 10% of all foster parents worked as professionals, the remaining 90% received an honorarium of 15,000 HUF a month per child, which was seen as a symbolic gesture of thanks, rather than a wage. This means that now the majority of foster parents receive more remuneration than they had before they were "professionalized". They are now covered by the social security system, and the years spent fostering count toward their old age pensions. Yet the fact that they are getting wages below the national minimum for extremely taxing work that takes up every second of their lives is not missed by foster parents. As one of them succinctly put it: "If you insist on calling this paid work, you might as well call it slave work" (Ilona, foster mother of a small boy in a rural town). In addition to the salary, each child receives an allowance from the state. This sum must be solely dedicated to his or her living costs, and, as I have already noted, foster parents must document the spending in detail. The size of the allowance is tied to the minimum old age pension payment (it amounts to roughly 150% of it). As I pointed out in the previous chapter, the level of the national minimum old age pension has not changed since 2008 and thus has been devalued by about 30% since 2010. Most foster parents and social workers agree that the cost of raising children is higher than the allowance, especially for older children or children with special needs. Some agencies allocate extra funds for foster parents for specifc costs (such as glasses or dental work) but these are unpredictable and vary across agencies. Similarly, foster parent network agencies occasionally distribute treats on special occasions, such as vouchers to buy gifts at Christmas or Easter—the spending of which has to be carefully documented. Nevertheless, several foster parents claimed that they supplement the cost of food and basic necessities from their own salaries or that they couldn't afford to foster if their husbands had not been making a decent salary. #### Increased Work Volume Many child protection experts agree that the job of fostering is getting harder and harder. As I pointed out earlier this argument was used to justify the need for disciplining, regulating, "professionalizing" foster parents. Here is another very experienced foster parent advisor describing the situation: Children come from increasingly diffcult life conditions. Their problems are often more complex and diffcult to deal with. So practically every single child would need a learning therapist, a psychologist, psychiatrist, a developmental therapist, a physician. What I mean is that life had been harder on these kids than for those who we had years ago, and to help them and treat this is no small task. (Foster parent advisor, social worker, Budapest agency) This is especially so because foster parents raise a growing number of children. The number of children in need has been increasing, yet national campaigns to recruit foster parents have not been particularly successful, so their numbers have been stagnating. In 2000, 25% of foster parents raised three children or more, in 2010 almost 40% did, and in 2020 the fgure stood at over 50%. Until 2000 more than half of all foster parents raised only one child. Now there are fewer than 20% working in this category (KSH 2019a). More children, especially more children with major psychological or developmental issues, means signifcantly more work per parent. This process started earlier, but the incentive structure set up by the 2014 regulation has reinforced it. It is making increasingly more sense to consider fostering as one's only paid job rather than as something to do in addition to working elsewhere. In this context, more children are needed to make a livable, even if meager, income. # Heightened Expectations, Surveillance and Control As foster parents' relationship to the state turns into one of employment, expectations on the part of the employers increase as well. The administrative burden has grown with the employment relationship. As a policy expert in the Child Protection Service explained to me: "We expect them to be more disciplined, more cooperative. This is in fact the goal of changing the relationship into a professional one. That and that they should be required to participate in trainings." Both the agencies and foster parents agreed with this claim. To illustrate I selected the words of a foster parent who compared the heightened expectations to the ridiculously low compensations she receives: Especially now that they [the state] put them [the legal guardians] on my shoulders too … sure I get some money in return. Now I get all of 30,000 HUF. And this should make me feel really good because I now have a salary and this will allow them to tell me what to do. (Viola, foster mother of two children) As already suggested in the above quote, in addition to an increased workload, heightened expectations and low wages, the process of professionalization is also accompanied by an intensifcation of surveillance over foster parents' work. I'd like to help children live in a happy, safe, well-balanced, normal family. But instead, I assign them [the foster parents] an external guardian, send a foster parent advisor on them, maybe more than one guardian because each child could have a different one. And I turn the home of foster parents into a zoo … who, by the way, I force to take the child to visitation meetings with birth parents every weekend. (Director of a foster parent network agency, Budapest) At least two supervisors visit families on a regular basis: the foster parent advisor is the most important and is the parent's primary contact to the agency. In addition, as of 2014 each child has a legal guardian, who is a representative of the state, and who makes all fnal decisions for the child. He or she also visits regularly and, as the quote suggests, guardians are assigned to children, not families, so a family may have several such offcials involved in their lives now. In addition, representatives from the agency may also stop by for a variety of reasons. Some of these visits are unannounced, but most are arranged in advance, depending on the schedules of the parties involved. Most foster parents had only a vague idea of how often exactly the visits are supposed to happen, but they did sense that they must accommodate someone almost weekly. This coupled with the bi-annual weekend trainings they must attend, network-wide holiday gatherings, as well as the bi-weekly or monthly birth parent visitation sessions, which also often happen at the agency's premises with supervision from the agency, all taken together provide a great deal of opportunities for contact. Typically, foster parents try to build a good relationship with their advisors as they see them not only as their direct supervisors but also as their contact to the agency. Advisors also serve as a source of practical advice or emotional support. During their monthly visits, the foster parent advisor talks to the child and the foster parent, but also has the right to open refrigerators, wardrobes and toy storage boxes to make sure the child has all that is prescribed by law and deemed necessary by the agency. They also assess the cleanliness of the home in general, and comment on it should they fnd it not up to their standards. I am being continuously monitored. When Adam came to live with us, they [representatives of the foster parent network agency] visited me twice a week. I cleaned more than ever, because I was worried that they would fnd something amiss. Then, after a while I got used to the visits and gave up on the extra cleaning. (Natalia, foster mother to newborns, living in a small town) Natalia is a relatively recent foster parent who specializes in looking after newborns until they get adopted. She has a college degree, but gave up her job as a marketing manager a few years ago, and now lives with her husband, three school-age birth children, and a varying number of fostered babies in a small town about 60 minutes east of Budapest. She is perhaps the most vocal about the ongoing surveillance, but several foster parents told me horror stories they had heard of especially brutal advisors, for example the one who stopped by randomly during the weekend lunch period and looked into pots to see what the children were being fed. The stories may not be true, but they do refect foster parents' understanding of their vulnerability to the gaze of their advisors, which penetrates even the walls of their bedrooms. Their lack of control over their work lives manifests most often when they are assigned children or when children leave their homes. Several accounts describe how foster parents are increasingly unable to infuence these two vital processes. One foster parent, for example, was asked to take in three children with exactly two days' notice. She had space for two children, but had no control over either the number or the timing of their arrival. Once they identify a child they ask you to host, they call you on the phone. In principle you would have time to discuss this with your family and give an answer in a few days. Yeah, dream on. I'll tell you how these three children landed here and you'll see. When we were just receiving an honorarium we could say no. Then we had this training and they told us that we'd better agree to accepting the children they send us. (Anikó, foster parent of four children, rural town) Foster parents are especially prohibited to reject children on the basis of ethnicity. This is an important issue, since—according to estimates between 30% and 50% of the children in the system are of Roma ethnicity and, as I explained above, discrimination against the Roma are widespread. Foster parents are not immune to racism either, even though they are trained explicitly to avoid it. Some express fear of how the child will be accepted in their local communities, others are concerned about meetings with the birth parents. Yet others simply feel animosity toward a child who looks "different". Thus some of the prohibition against picking and choosing of children has to do with the agency's fully justifable desire to avoid racial discrimination. But the point here is that foster parents noted a change in attitude toward them since the start of their employment relationship and argued that they became more vulnerable as a result of the new form of dependency. For their part, foster parent advisors are well aware of their role in the system. I try to stay friendly with them [the foster parents] so they wouldn't see the supervisor in me but the helper. But obviously my main role is to follow up on whether or not their work serves the interests of the child. But I am usually friendly with them and I do the checking up part while we are chatting and I help a great deal if needed. (Emese, foster parent advisor, rural town) Until 2014 most foster parents had legal guardianship rights over their children, but in 2014 each child was assigned a separate guardian, and foster parents could not take on this role any more. This was experienced as a logistical hurdle, but also an expression of lack of trust and loss of control. One [problem with the new situation] is that they don't trust us [to make the right decisions for the child].The other is that we have another person who we are accountable to. (Marika, long-time foster parent, now raising four children) Or as another experienced foster parent explained why she found it offensive that legal guardians now have the fnal word on major life decisions about the child: You know, it is really strange [to have a guardian overseeing her work]. I am raising this child. I know what he needs. I am responsible for him too. Yet, I don't make the decision, I am sometimes not even asked. (Paula, foster parent to two children) #### Gendered Altruism as a Form of Resistance Consistent with the logic of carefare, work conditions have worsened and wages are appallingly low. Why do foster parents agree to these employment conditions? One possible answer is that they simply have no alternatives: the "whip of hunger" forces them to accept even these conditions. But this is inconsistent with what I heard from my interviewees. Most foster mothers I talked to could list several job alternatives, or positions they gave up for fostering. Granted, some of those jobs required long traveling, shift work or working very long hours, but they did not necessarily mean more overall effort than the work they were doing now. Several of my respondents said that they chose fostering because they wanted to help children, and associated caring for others with their true feminine identity. In other words, they evoked altruism and the importance of a meaningful, caring life as a form of highly gendered moral rationality (Duncan and Edwards 1999), that is, as a rational choice, which was not based on economic gain but on a specifc orientation to life and a system of values sharply at odds with the mainstream expectations. A good example is Zsuzsa, an engineer, one of the few foster mothers I interviewed who had a professional, full-time job in addition to raising two fostered and two biological children (with a stay-at-home husband). She told me that her friends and family do not quite understand why she and her husband chose to foster on top of all her other work. But, she said, "We wanted to do something meaningful, something that we can later explain to our children" (Zsuzsa, fostering two children, biological mom to two in Budapest). Blanka, who used to work as a nurse in a nearby hospital before she resigned to raise foster children explained to me laughing: "I know this sound silly but I don't like to be working for money" (Blanka, foster mom to 2 toddlers, small rural town). As a corollary, foster parents often claimed that what they did was not work but part of the natural fow of their lives, part of who they were. One example of this position comes from Tanya, a long-time foster parent who lives in a village outside of Budapest. She acknowledges that fostering requires energy, but makes a sharp distinction between work and family. The term "work" has no place in the "natural" setting of a family: I don't think of this as work. Because I think this is a natural thing. We have children, we come home, just like raising my own kids, this is not work either. I mean there's a lot of work with this, yes, and I get really tired by the evening … but this is not a job for me. It is family. (Tanya, foster mother of three) Other women claim that what they do does not require special skills, only their womanly instincts. Kata, a foster mother of two with two biological children of her own said this: I am not trained as a nurse, I cannot offer anything extra. I am just a mommy who is simply capable of raising healthy children. She is seconded by another foster parent who, when I asked her if she considered what she did as work, replied: "I … I don't really know. To me this is really routine, not a big deal at all. No, this is not work" (Juli, longtime foster mother, currently raising three children). An important correlate of altruism is that love seems incongruent with fnancial compensation: both foster parents and their agencies subscribe to the "hostile worlds" argument (Zelitzer 1997). This was one of the reasons why I heard repeatedly that fostering should not be thought of as paid work. Instead, foster parents and their advisors used a variety of different words to describe what they were doing including "love", "calling", a "hobby", a "lifestyle", a "way of life", a "service": fostering was thought of as outside the realm of paid work as something opposite to the world of work and fnancial compensation. If I were a child, I wouldn't want my foster mother to get paid so she would love me and keep me in her family. I don't think that would be good, not even for the self-resect of foster parents. With this move [the 2014 legislation] they took away the only important thing, that they can do charitable activity, that they can help kids. Now we say this is their job, like for a teacher or child care worker. (Diana, experienced foster parent advisor in Budapest) Or as another policy expert put it: This is all about children, we cannot treat this as work. A foster parent must be a lot more than that. It is not enough just to satisfy the daily needs of the child, to offer clean clothes, room and warm food. An institution can offer all that. A foster parent needs to give more: her soul and her love. (Social worker, policy expert) Soul and love cannot be bought and, it seems, need not be compensated for either. # Conclusion This chapter illustrated an important yet well-hidden aspect of Hungary's carefare regime: the construction of primarily female, paid—but barely so—care workers through the process of professionalization from above. A new piece of legislation, fully in line with expert recommendations, transformed the relationship of foster parents to the state into that of employment. As employment contracts go, this one is quite unfavorable to foster parents: it increases their work volume, the expectations placed on them and the length of training required for the work, it exacerbates the surveillance foster families are subjected to by state actors, and wrests from them even more control over the work process. In exchange for work done in these extremely precarious conditions, foster parents get paid less than the minimum wage. They also became part of the social insurance scheme and now expect to draw pension benefts—proportionate to these wages. Foster parents thus joined the underclass of female care workers whose exploitation forms one of the foundations of the anti-liberal regime. The transformation in this area of child protection also highlights the reengineering of the state in an anti-liberal political direction: the channeling of public funds into the coffers of politically loyal religious organizations. Both the discursive justifcation of the changes in state policies, and foster parents' resistance to these, are based on the assumption that caring is women's natural skill and the ultimate meaning of their lives. State agencies can afford to pay precious little for the vast amount of work foster parents do, and foster parents can justify their acceptance of these extremely precarious work conditions by relying on a gendered hostile world argument: love for children and proper fnancial compensations do not mingle, and women's ultimate expression of femininity is in the work of caring selfessly for others. As long as foster parents, especially foster mothers, agree to conceptualizing their employment relationship as one of motherly altruism, and feminine meaning making, carefare will thrive. # References Bátory, Ágnes. 2016. Defying the Commission: Creative Compliance and Respect for the Rule of Law. Public Administration 94 (3): 685–699. Abbott, Andrew D. 1988. The System of Professions: An Essay on the Division of Expert Labor. Chicago, IL: University of Chicago Press. ———. 2019b. Neveloszülo ̋ k [Foster Parents] ̋ . Budapest: KSH. https://www.parlament.hu/documents/10181/1789217/Infojegyzet\_2019\_34\_neveloszulok.pdf/c2a1487e-3ff0-eb07-3a20-13851d22e8fb. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Conclusion Abstract This chapter is a brief summary of the main arguments and a development of the point that carefare policies form an integral part of the political success and legitimacy of the anti-liberal regime. The chapter also shows how selective and exclusionary the policies are. Keywords Anti-liberal • Hungary • Gender inequality • Class inequality • Carefare "God created men and women so they together may form a whole, which when complemented by children—we call a family. In the family and in society women embody gentleness, devotion, care, empathy, beauty, complementing men so they together can show the way to the next generation. Mothers are the heart and soul of families, the whole society and the nation." (Offcial Facebook post by MiklósKásler, Minister of Human Resources, March 7, 2021) To mark International Women's Day in 2021, the European Commission issued a statement which enumerated women's multiple contributions to the fght against the COVID-19 pandemic: their work as doctors, nurses, teachers and shop assistants. The communiqué emphasized the European Union's commitment to gender equality and listed a range of measures to be introduced in the near future to this effect.1 The Hungarian Minister of Human Resources, for his part, took a rather different angle. In a statement on his Facebook page quoted at the top of this chapter, he chose to greet heterosexual mothers only on International Women's Day and laud them for their gentleness, caring and kindness. He emphasized the fact that women's role was to complement men's work and reminded us that women's contributions to families, society and nation all belong to the realms of the heart rather than to the material world. The contrast between the two approaches—the European Commission's and the Hungarian government's—could not be sharper and could not illustrate more clearly the main arguments in this book. A new type of political order has emerged in Hungary since 2010: a form of authoritarian capitalism with an anti-liberal political and social agenda. An important part of this agenda directly targets gender relations, specifcally women and women's work. To conclude and summarize the points I made in the previous chapters, let me start with Gabor Scheiring's (2019: 254) analysis of social and political developments in Hungary. Scheiring calls the post 2010 Hungarian state "accumulative" and shows how FIDESZ and its cadres are deeply involved in reshuffing the existing class structure: they are creating their own politically loyal and economically powerful bourgeoisie through the process of state-assisted, dubiously legal, capital accumulation. The expropriation of resources, however, is taking place at the expense of those in the lower half of the social hierarchy. Scheiring points out that this is bound to lead to polarization and social tension, which may explain the authoritarian turn in Hungarian politics. Authoritarian rule, specifcally authoritarian populist strategies are being deployed to retain the support of the economically disadvantaged. The government's discursive construction of "moral panics" successfully transforms conficts about widening economic inequalities into disagreements about cultural and ideological issues (ibid.). Indeed, one key example of such a moral panic has been the government's purported struggle against what they call the international "gender lobby". As I pointed out <sup>1</sup> See Statement by the European Commission ahead of the International Women's Day 2021. https://ec.europa.eu/commission/presscorner/detail/en/statement\_21\_890. Numerous critiques question the EU's commitment to and limited conceptualization of gender equality (see, e.g., Repo 2016). The point here is the contrast between the messages directed at women in Hungary and in the European Commission only. in Chap. 1, this has been sustained, if with slightly modifed content, by the government-friendly media for the past four years.2 Starting in the mid-2010s Hungary's anti-liberal political regime began to deploy an even more spectacular strategy to ameliorate social confict around redistribution: pronatalist family policies. The decrees passed from 2014 onward provide a signifcant amount of cash support—some earmarked for specifc purposes, others freely usable—to families with children. Better-off families receive and can utilize a larger share of the subsidies but even lower-class, working families are able to access several of the newly introduced tax credits, baby loans and mortgages. This may open new fnancial possibilities for eligible families among the roughly one-third of Hungarians who had been unable to set money aside as savings and for whom investment in housing, for example, may have seemed like a hopeless goal. In other words, the fnancial rewards of the newly introduced family policies reach social groups in the lower half of the social hierarchy, many of whom had been losing hope when faced with the diffculties of fnding decent, stable jobs which pay a living wage, and the sluggishness of the rate of intergenerational upward mobility (Huszár et al. 2020). The "family protection measures" of the recent Orbán government guarantee that some limited resources trickle down to this group, who are a crucial part of FIDESZ's electoral base (Róna et al. 2020). The process does not ameliorate class inequalities because of the highly selective targeting of the rewards of family policies, but the measures have, nevertheless, impacted and partially reorganized not only gender relations but also the stratifcation order. Hungary's anti-liberal government has utilized the re-regulation of gender relations to modify socio-economic inequalities in a politically effcacious way. 2The most recent variety of the "gender panic" at the time of writing this chapter is related to homosexuality and the supposed threat it poses to the future of the Hungarian nation, Christian civilization and/or the morality of children. In the frst three months of 2021, Magyar Nemzet [Hungarian Nation], the vehemently pro-government online daily I described in Chap. 1, published 79 articles containing the term "LGBTQ". (Of the roughly similar number of articles on "gender" in the same period almost half contained the term LGBTQ and many more lamented the threat of non-heterosexual forms of sexuality without explicit reference to the term.) Nothing proves better the fact that this is a "moral panic" artifcially created for political reasons is the admission of the Hungarian Minister of Family Affairs, Katalin Novák, herself who pointed out that issues related to non-heterosexuality, specifcally gay adoptive parents, is not among the "top 100 problems of Hungarian people" (February 20, 2021). It is nevertheless kept on the political agenda by the governmentcontrolled media. The principle of "divide and conquer" is part of this political strategy. Not everyone is eligible for the tax benefts or the child allowances. First, the vast majority of funding is tied to sustained participation in the formal paid labor market, and more than one income within the family which is above the national minimum. In 2018, 73% of the working age (20–64 years old) population was employed for wages. Although employment rates have increased most among the least educated social groups in recent years, vast inequalities among the Roma ethnic minority and the non-Roma majority remain: in 2018 the employment rate of Roma men and women was 44% and 23%, respectively, compared to the employment rates of 81% for non-Roma men and 65% for non-Roma women. Most of the Roma population are thus simply excluded from the government windfall, notwithstanding the fact that they are more likely to have children than the majority of the population and certainly are more likely to be in need of support in order to move out of poverty. In addition, according to recent estimates, close to 40% of all employees are contracted to work for the minimum wage, reducing the possibility for other groups to partake of the newly available resources. Second, even among those who are employed and fnancially eligible, the majority do not have children in their households: about 22% of those employed have one child, 17% two children and only 6% have more than two (Bakó and Mészáros 2019). This means that only about 40% of the population between 20 and 64 years of age are eligible to apply for the subsidies which are tied to both labor market participation and the presence—or promise—of children. It is these white, heterosexual, working families—constructed as "deserving" by mainstream political propaganda—who are targeted by the recently passed measures. The benefts received via the wide range of pronatalist policies and related measures do not guarantee long-term compensation for raising children. However, in the late 2010s, when paid work was plentiful, the primarily one-off benefts allowed a respite, offered new opportunities to buy a larger house, to renovate a home and to spend a little more money on necessities, which might not have been possible otherwise. In addition, those at the bottom of the income distribution saw a proportionately larger increase in their wages, thanks to the tax credits, than those at or above the average.3 This potential upward mobility is quite fragile: in the <sup>3</sup> If you are a member of the "deserving" families and you are employed and make the minimum wage and together with a spouse can claim tax credits for two children, your joint case of someone losing their job they also lose eligibility, yet loans still need to be paid back. A divorce—not a rare occurrence in Hungary—may leave women especially vulnerable, and a variety of life events may prevent a family from having the number of children they had promised when they signed up for the government's loan. The positive impact of these policies may last until the next election in 2022 but the risks involved for individual people are numerous. Anti-liberal rule is thus built on the backs of women, especially on the backs of hard-working, ambitious, lower-class women. Women's work burden is likely to increase if families are to access the tax credits, the baby loans, the cheap mortgage and other subsidies. The funding is available on condition that they have more children, and—given the typical division of labor within households—take on more care responsibilities, dedicate more time to care work. At the same time, they will continue working for wages too. But employers in the Hungarian labor market—both state and private, domestic and international—operate by regulations which largely ignore care responsibilities and thus disadvantage women. Alternatively and increasingly, the carefare state offers job opportunities in the care industry specifcally for mothers—such as fostering as Chap. 3 demonstrated—with the typically appalling work conditions that approximate those available to live-in migrant care workers in other parts of the world. Should women accept these conditions, their families may access these precarious "gifts" endowed by the government, but should they reject any parts thereof, they immediately become second-class social citizens. Furthermore, the participation of eligible women and families in the programs lends legitimacy to the government policies' singling out and constructing the "deserving", working, heterosexual family with children as the social group responsible for the country's future and the only true hope for it. I have called this set of policies "carefare". Carefare policies, like workfare or prisonfare, are designed to discipline vulnerable workers into doing net monthly salary increases from 220,000 HUF to 260,000 HUF, which is an increase of 18%. But if both you and your spouse make the average wage and receive 560,000 HUF a month, the additional 40,000 HUF tax credit for two children amounts to a mere 7% of your income. In other words, those at the bottom receive a higher increase relative to their wages through the child tax credit system. Similarly, a "baby loan" of 10 million HUF is of signifcantly more value to those at the bottom of the social hierarchy than to those whose regular income is higher. If, however, low-income families do not produce the required three children, the burden of paying back the loan is also much heavier. vastly undervalued work in exchange for claiming their social citizenship rights. Even though its elements are familiar from welfare policies elsewhere, Hungary's carefare regime represents a novel response to what has been called the care crisis emerging in fnancialized global capitalist economies: the confict between the intensifcation of claims made on workers' energies by their jobs and the simultaneous necessity to provide intensive parenting to children and care for others in need in local communities (Fraser 2016). Hungary's anti-liberal government has not fully resolved the confict but it is experimenting with a new solution. The incentive structures of carefare policies, government propaganda which sentimentalizes women's work and sets it in contrast to remuneration, the lack of feasible and enforceable gender equality measures and economic alternatives, and the non-existence of woman-friendly trade union or women's rights movements all work together to force women into having to increase their work burden and accept inferior and thus more vulnerable positions in the paid labor market. This book has described Hungary's gender regime. There may be an elective affnity between anti-liberalism and pronatalist "family protection" policies or the rejection of the principles of gender equality, but the relationship is certainly not deterministic or causal. Hungary's geopolitical position, its economic dependence on foreign investors and EU structural funds, its history of failed modernization projects tied to a variety of women's emancipation agendas, the underdevelopment of democratic institutions, the recent trauma of social upheaval and numerous economic crises, as well as the history of authoritarian leaders and centralized propaganda, all add up to a confuence of conditions that lead to unique outcomes. Anti-liberal, authoritarian-leaning political rule is spreading fast in every part of the world; thus Hungary's carefare regime should serve, if nothing else, as a framework to guide future comparative work on gender, social citizenship rights and the conditions of paid and unpaid work. # References Bakó, Tamás, and Judit Mészáros. 2019. A magyarországi munkaerőpiac 2018 ban [The Hungarian Labor Market in 2018]. In Munkaeropiaci Tükör [Labor ̋ Market Report], ed. Károly Fazekas, Márton Csillag, Zoltán Hermann, and Ágota Scharle, 15/31. Budapest: Közgazdaság- és Regionális Tudományi Kutatóközpont. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Appendix #### List of major "family protection" policies, Hungary 2021 (continued) #### (continued) (continued) (continued) #### 114 APPENDIX # Index1 #### A Abortion, 34, 35 Altruism, 98–100 Anti-gender, 1–22, 54 Anti-immigration, 36 Anti-liberal, 1–10, 13–16, 20, 30–32, 37, 43, 47, 56, 57, 65, 70, 73, 78, 100, 104, 105, 107, 108 #### B Baby loan, 105, 107, 107n3 Birth, 11, 12, 33, 34, 39–41, 44–46, 54–56, 68, 70, 78, 79, 82–88, 90, 95–97 #### C Care work, 3, 10, 12, 14, 16, 31–33, 37, 39–42, 44–47, 51, 52, 54–58, 65, 76, 107 Carefare, 1–3, 16, 20–22, 29–58, 65–100, 107, 108 Central and Eastern Europe, 12 Childcare, 9, 13, 39, 44, 45, 50, 53, 68, 77, 87, 91 Child protection, 21, 22, 66, 68–73, 75–77, 93, 100 Children's home, 67, 69 Church, 7, 43, 72–75 Churchify/churchifcation, 41–43 Class inequality, 3, 32, 57, 105 Commodifcation, 41, 72 COVID-19, 45, 51, 103 Crisis of care, 3, 30, 33, 36, 41, 56 #### D Demographic crisis, 33–34, 36 Deregulation, 41, 43, 72 Deserving family, 32, 57, 106n3 Disinvestment, 41–43 Divorce, 12, 40, 58, 107 1Note: Page numbers followed by 'n' refer to notes. © The Author(s) 2022 115 E. Fodor, The Gender Regime of Anti-Liberal Hungary, https://doi.org/10.1007/978-3-030-85312-9 #### E Earned income tax credit/tax credit, 39, 40, 42, 44, 105–107, 106–107n3 Emotional work, 79–86, 91 Employment, 8, 9, 11, 32, 37–42, 46, 48–51, 66, 69, 70, 75–77, 79, 91, 94, 97, 98, 100 Employment rate, 48, 51, 106 European Union (EU), 4–9, 11, 14, 18–20, 32–36, 42, 45, 48, 52–54, 57, 68, 73, 92n2, 103, 104n1, 108 Exploitation, 15, 44, 49, 65, 100 # F Familialization/familialism, 44, 45 Family beneft, 38–40, 50, 51 Family friendly, 50, 52, 75 Family protection measure, 57, 105 Father, 13, 45, 51, 83 Femininity, 54, 56, 100 Fertility/fertility rate, 12, 33, 35 Foster home, 66, 69, 79, 83 Fostering, 65–100, 107 Foster parent, 21, 22, 66, 68–70, 73–100, 92n2 Foster parent advisor, 70, 76, 81, 85–87, 89–91, 93–95, 97, 99 Foster parent network/foster parent network agency, 22, 70, 73, 74, ### G Gender equality, 2, 3, 14, 15, 20, 32, 47, 54, 103, 104n1, 108 Gender equality policy, 14–16, 20, 54–56 Gender ideology, 15, 16, 19, 20 81, 88, 93, 95, 96 Gender inequality, 3, 15, 16, 18, 20, 47, 49 Gender regime, 1–3, 5, 10–16, 30, 108 Grandparent, 40, 41, 45, 46 Guardian, 70, 76, 86, 88, 90, 94, 95, 97 GYED/GYES, 92 # H Heterosexual, 32, 39, 42, 56, 104, 106, 107 Hostile worlds, 99, 100 # I Illiberal, 4, 5, 15 Informal work, 39, 50 # L Labor control, 77, 78 Labor market, 3, 8, 11, 12, 16, 18, 34, 47, 48, 50, 51, 54, 56, 57, 65, 77, 92, 106–108 # M Male breadwinner, 47 Materialist welfare state, 36 Maternalist welfare state, 36 Migrant, 18, 19, 36, 107 Migration, 18, 19, 36 Minimum wage, 8, 9, 38, 40, 50, 92, 92n2, 100, 106, 106n3 Mother, 12, 13, 21, 37, 38, 40, 41, 45–47, 51–53, 55, 78, 83–88, 90, 91, 93, 94, 96, 98–100, 103, 104, 107 Motherhood penalty, 21, 47, 51–53 Multigenerational, 12, 40, 46, 51 #### N Nation, 19, 20, 33–35, 37, 46, 103, 104, 105n2 Nationalism, 15 Neoliberal capitalism, 4, 8, 30, 33 Neoliberalism, 15, 41 Nursery school, 43, 72 #### O Orbán regime/government, 8, 16, 35, 42, 56, 57, 68, 105 #### P Paid work/waged work, 3, 10–14, 32, 38–40, 44–48, 50, 55, 67, 93, 99, 106, 108 Parent, 9, 12, 31, 38–41, 46, 53, 78–80, 82–86, 88, 90, 91, 94, 95, 97, 105n2 Parental leave, 38, 40, 44–46, 51, 53, 92 Part-time, 50, 52, 53, 88 Population, 9, 12, 13, 20, 31, 33–36, 38, 41, 51, 57, 86, 106 Poverty rate, 57 Precarious, 47, 50, 100, 107 Professional, 4, 7, 9, 21, 53, 68, 74–77, 85–86, 93, 94, 98 Professionalization, 75–78, 95, 100 Pronatalism, 3, 10 Public works, 9–10, 42, 51 #### R Refugee, 35, 36 Reproductive work, 12, 44 Roma, 13, 86, 89, 96, 106 #### S Sentimentalization, 47, 54–56 Smart love, 85 Social citizenship, 10, 31, 36–41, 56, 108 Social insurance, 31, 37, 38, 100 Social policy, 20, 30, 31, 35, 36, 41 Social protection, 41, 42 Social reproduction, 30, 32 State expenditure, 71 State socialism, 11, 12, 33, 36, 43, 50 Surveillance, 76, 77, 94–97, 100 #### T Tax credit, 39, 40, 42, 44, 105–107, 106–107n3 #### U Underclass, 47–51, 57, 66, 76, 91, 100 Universal right, 38–39 #### W Wage gap, 11, 49 Wages, 8, 9, 11, 13, 32, 38, 39, 41, 44, 46–57, 66, 75, 76, 89, 91–93, 95, 98, 100, 105–107, 106–107n3 Welfare regime, 41 Welfare typology, 45 Workfare, 3, 41, 42, 50, 107 Work hours, 9, 13, 21, 51, 52, 76, 80 Working class, 32 Work-life balance, 32, 47, 51, 52	doab	2025-04-07T03:56:59.037116	14-12-2021 05:01	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "006db864-879f-4bae-9f57-b9605ac62633", "url": "https://library.oapen.org/bitstream/20.500.12657/51949/1/978-3-030-85312-9.pdf", "author": "Fodor, Eva", "title": "The Gender Regime of Anti-Liberal Hungary", "publisher": "Springer Nature", "isbn": "", "section_idx": 0 }
006df1b2-f518-4d6b-8d96-484202f13086.0	Edited by Fahmina Zafar and Anujit Ghosal To ensure a healthy lifestyle, fire safety and protocols are essential. The population boom, economic crunches, and excessive exploitation of nature have enhanced the possibilities of destruction due to an event of a fire. Computational simulations enacting case studies and incorporation of fire safety protocols in daily routines can help in avoiding such mishaps. ISBN 978-1-83962-425-4 Fire Safety and Management Awareness Published in London, UK © 2020 IntechOpen © stockphoto / iStock	doab	2025-04-07T03:56:59.067269	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 0 }
006df1b2-f518-4d6b-8d96-484202f13086.1	Fire Safety and Management Awareness Edited by Fahmina Zafar and Anujit Ghosal	doab	2025-04-07T03:56:59.067388	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 1 }
006df1b2-f518-4d6b-8d96-484202f13086.2	Fire Safety and Management Awareness Edited by Fahmina Zafar and Anujit Ghosal Published in London, United Kingdom ## Supporting open minds since 2005 Fire Safety and Management Awareness http://dx.doi.org/10.5772/intechopen.87859 Edited by Fahmina Zafar and Anujit Ghosal #### Contributors Rositsa Velichkova, Ivan Antonov, Svetlin Antonov, Kamen Grozdanov, Vancho Adjiski, Zoran Despodov, Rebecca Abney, Qin Ma, Agnes Iringova, Brady Manescau, Khaled Chetehouna, Aijuan Wang, Ilyas Sellami, Anujit Ghosal, Fahmina Zafar, Quentin Serra, Eric Florentin, Rachid Nait-Said, Fatiha Zidani #### © The Editor(s) and the Author(s) 2020 The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be found at http://www.intechopen.com/copyright-policy.html. #### Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in London, United Kingdom, 2020 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, 5 Princes Gate Court, London, SW7 2QJ, United Kingdom Printed in Croatia British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Additional hard and PDF copies can be obtained from [email protected] Fire Safety and Management Awareness Edited by Fahmina Zafar and Anujit Ghosal p. cm. Print ISBN 978-1-83962-425-4 Online ISBN 978-1-83962-426-1 eBook (PDF) ISBN 978-1-83962-427-8	doab	2025-04-07T03:56:59.067423	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 2 }
006df1b2-f518-4d6b-8d96-484202f13086.3	We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists 5,100+ Open access books available 126,000+ International authors and editors 145M+ Downloads 151 Countries delivered to Our authors are among the Top 1% most cited scientists 12.2% Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) ## Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com	doab	2025-04-07T03:56:59.067863	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 3 }
006df1b2-f518-4d6b-8d96-484202f13086.4	Meet the editors Dr. Fahmina Zafar is a senior researcher working at the Department of Chemistry, JMI, New Delhi, India, under the Women Scientists Scheme for Research in Basic/Applied Sciences, DST, India. Dr. Zafar received her PhD (2006) and MSc (1999) degree in Chemistry from JMI. She has worked as a postdoctoral fellow under UGC Kothari Postdoctoral Fellowship, and as a Scientist Pool, Research Associate, and Senior Research Fellow (CSIR) at the same department. She has more than 70 publications in peer-reviewed journals and books, and has presented more than 40 research papers in national and international conferences. Her research work involves the development of bio-based polymers, metallopolymers, organic-inorganic hybrids, coordination polymers, and nanocomposites for green environment in different fields including adsorption, antimicrobial, and corrosion-protective applications. Dr. Anujit Ghosal is currently working as a young researcher at the School of Life Sciences, Beijing Institute of Technology, Beijing, China. He received his PhD degree (Chemistry) in 2015 and his M.Sc. degree (Materials Chemistry) in 2009 from Jamia Millia Islamia (J.M.I.), New Delhi, India, as well as his BSc degree with honours in Chemistry from Zakir Husain Delhi College, University of Delhi, Delhi, India in 2007. He has worked as a National Postdoctoral Fellow under a SERB fellowship in the School of Biotechnology, Jawaharlal Nehru University, New Delhi, India and served as an Assistant Professor for one year in Galgotias University, Greater Noida, UP, India. He has published in various international reputed refereed journals and participated in many national/international workshops. His research expertise involves polymer chemistry, hydrogels, magnetic nanomaterials, dye adsorption, drug delivery, and electrochemical corrosion. Contents Section 1 Section 2 Section 3 Preface XI Introduction 1 Chapter 1 3 Effect of Variable Parameters 9 Chapter 2 11 Chapter 3 31 Advanced Protection Mechanism: Simulations 45 Chapter 4 47 Chapter 5 71 Introductory Chapter: Fire Prevention Strategies Numerical Study on the Outdoor Wind Effects on Movement Shifting Wildfire Trends and Management Implications for the Mathematical Modeling and Simulation of Development of the Methodology for Optimal Fire Evacuations in Underground by Brady Manescau, Khaled Chetehouna, Quentin Serra, Wildland Urban Interface in the Twenty-first Century by Ivan Antonov, Rositsa Velichkova, Svetlin Antonov by Anujit Ghosal and Fahmina Zafar Smoke along a Corridor Aijuan Wang and Eric Florentin by Rebecca Abney and Qin Ma Fires in Confined Spaces and Kamen Grozdanov Mines Based on Simulated Scenarios by Vancho Adjiski and Zoran Despodov ## Contents Preface An important saying is "precaution is better than cure". The awareness of past and present adverse situations caused by fire outbreaks that have and are affecting living organisms directly or indirectly is mandatory for developing future preventive measures. Fire is considered to be a natural calamity, but in recent years it is most likely caused by human error. Awareness and improvement in prevention protocols is of prime importance for sustainability. Human development and other unplanned activities have further increased fire tragedies. The increased population and economic burden have also led to the construction of more confined and populated buildings, small utility spaces as well as narrow exit routes. Literature surveys and case studies are symbolic proof of the destructive ability of man-made or natural fire eruptions worldwide. Advanced fire or smoke sensors, use of particular construction material, evacuation plans for a sudden fire, installation of fire protection measures, and study of previous fire incidents are the keys to establishing awareness among professional and everyday people. In this regard, computation modelling and simulations have helped the analysts to prepare future housing projects or study previous fatal events in great depth. Under fixed parameters, recent technological advancements have made it possible to analyze and rectify the past or probable future fatal events This book comprises seven chapters discussing few important aspects helpful in establishing safety against accidental fires and promoting awareness against them. The book discusses the improved construction strategies and ventilation designed to prevent accidents during emergency fires. Evacuation strategies and effects of external parameters (outdoor wind) on smoke are discussed using specific case modelling. Specific past events caused by fire have also been studied using numerical modelling to understand the disastrous scenario and improve the precautionary methods. This book will be helpful to students, researchers, and other related professionals The efforts of all the authors, contributors, and technical staff of IntechOpen, with special thanks to Ms. Dajana Pemac, Author Service Manager, have greatly helped > Fahmina Zafar Jamia Millia Islamia, > > Anujit Ghosal Beijing Institute of Technology, India China working in the field of fire prevention or management sections. caused by unwanted fire eruptions. in finalizing the book compilation. Rachid Nait-Said and Fatiha Zidani	doab	2025-04-07T03:56:59.067937	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 4 }
006df1b2-f518-4d6b-8d96-484202f13086.5	Preface An important saying is "precaution is better than cure". The awareness of past and present adverse situations caused by fire outbreaks that have and are affecting living organisms directly or indirectly is mandatory for developing future preventive measures. Fire is considered to be a natural calamity, but in recent years it is most likely caused by human error. Awareness and improvement in prevention protocols is of prime importance for sustainability. Human development and other unplanned activities have further increased fire tragedies. The increased population and economic burden have also led to the construction of more confined and populated buildings, small utility spaces as well as narrow exit routes. Literature surveys and case studies are symbolic proof of the destructive ability of man-made or natural fire eruptions worldwide. Advanced fire or smoke sensors, use of particular construction material, evacuation plans for a sudden fire, installation of fire protection measures, and study of previous fire incidents are the keys to establishing awareness among professional and everyday people. In this regard, computation modelling and simulations have helped the analysts to prepare future housing projects or study previous fatal events in great depth. Under fixed parameters, recent technological advancements have made it possible to analyze and rectify the past or probable future fatal events caused by unwanted fire eruptions. This book comprises seven chapters discussing few important aspects helpful in establishing safety against accidental fires and promoting awareness against them. The book discusses the improved construction strategies and ventilation designed to prevent accidents during emergency fires. Evacuation strategies and effects of external parameters (outdoor wind) on smoke are discussed using specific case modelling. Specific past events caused by fire have also been studied using numerical modelling to understand the disastrous scenario and improve the precautionary methods. This book will be helpful to students, researchers, and other related professionals working in the field of fire prevention or management sections. The efforts of all the authors, contributors, and technical staff of IntechOpen, with special thanks to Ms. Dajana Pemac, Author Service Manager, have greatly helped in finalizing the book compilation. > Fahmina Zafar Jamia Millia Islamia, India Anujit Ghosal Beijing Institute of Technology, China 1 Section 1 Introduction Section 1 Introduction 3 Chapter 1 1. Brief introduction Introductory Chapter: Fire A few decades back, the incidents of fire were regarded as natural calamity and were treated as such with very little thought on other aspects. However, in-depth analysis of most of the recent accidental fire tragedies indicates the human fingerprints such as deforestation, cigarette, firecrackers, static fire in forest at night or other similar incidences [1–3]. The frequency and extent of loss in life or economic damages have been increasing along with the population growth. So, to check the catastrophic misfortunate instances of fire and controlling the after-effects, stringent rules must be followed [4–6]. Broadly, the rules can be divided in three categories as shown in the Figure 1. In most of the places, fire safety and management protocols are common with slight modification based on the environmental conditions. These systems are adopted throughout the world, however, more strictly followed at flammable industries (oil, gas, paint, paper, etc.) while considered being little lenient at household vicinities [7, 8]. The increased population with constant high pressure on the economic backbone of numerous countries has altered the previous protection protocols [9–11]. The high cost of living has resulted in a more congested neighborhood i.e. population density particularly in the urban areas is constantly increasing [12, 13]. A collective approach is needed to avoid mistakes via., focusing on the fundamental area of management and planning. Figure 2 represents the basic blocks involved in planning and management section for any construction. Any small mishap such as an electric short circuit, heating system malfunction, barbeques, candles, or similar incidences at one place will affect the entire or adjacent community other than environmental contamination. Apart from the prevention of fire incidence, defined safety measures can save lives after the occurrence of such accidental events. Various approaches have been followed for the management of fire vi.Avoid cluttering the areas which can inhibit the fire extinguish mechanisms. vii.Recording alteration, submission, and safekeeping of building/factory preventions at the fundamental level. A few of them are presented below: ii.Routine inspections of doorways and exit passages iv.Maintenance of sprinklers or other extinguishers v.Keep track of the exact number of occupants iii.Conducting regular practice drills for fire evacuations i.Planning escape route blueprints Prevention Strategies Anujit Ghosal and Fahmina Zafar #### Chapter 1 ## Introductory Chapter: Fire Prevention Strategies Anujit Ghosal and Fahmina Zafar #### 1. Brief introduction A few decades back, the incidents of fire were regarded as natural calamity and were treated as such with very little thought on other aspects. However, in-depth analysis of most of the recent accidental fire tragedies indicates the human fingerprints such as deforestation, cigarette, firecrackers, static fire in forest at night or other similar incidences [1–3]. The frequency and extent of loss in life or economic damages have been increasing along with the population growth. So, to check the catastrophic misfortunate instances of fire and controlling the after-effects, stringent rules must be followed [4–6]. Broadly, the rules can be divided in three categories as shown in the Figure 1. In most of the places, fire safety and management protocols are common with slight modification based on the environmental conditions. These systems are adopted throughout the world, however, more strictly followed at flammable industries (oil, gas, paint, paper, etc.) while considered being little lenient at household vicinities [7, 8]. The increased population with constant high pressure on the economic backbone of numerous countries has altered the previous protection protocols [9–11]. The high cost of living has resulted in a more congested neighborhood i.e. population density particularly in the urban areas is constantly increasing [12, 13]. A collective approach is needed to avoid mistakes via., focusing on the fundamental area of management and planning. Figure 2 represents the basic blocks involved in planning and management section for any construction. Any small mishap such as an electric short circuit, heating system malfunction, barbeques, candles, or similar incidences at one place will affect the entire or adjacent community other than environmental contamination. Apart from the prevention of fire incidence, defined safety measures can save lives after the occurrence of such accidental events. Various approaches have been followed for the management of fire preventions at the fundamental level. A few of them are presented below: Figure 1. Three broad categories of the fire protection activities. #### Figure 2. Basics steps involved in fire protection of any construction. #### 2. Comprehensive views Although numerous prevention and fire safety manuals have been developed but with modernization redefining fire prevention protocols with time is mandatory [14–16]. However, the process of change is ever dynamic for the inclusion of new information. Incorporation of the output from upcoming research studies for avoiding miss-managements during a fire outburst and remodeling the evacuation strategies are important. The architectural improvements which can further assist the evacuation process and ease the smoke exhaustion process should always be considered. The new engineering strategies based on recent active research should be adopted in the construction of housing societies, particularly in highly populated areas [17, 18]. The implementation of fire safety protocol in pre-build structures 5 Introductory Chapter: Fire Prevention Strategies DOI: http://dx.doi.org/10.5772/intechopen.94037 causalities under such events. tion against tragedic incidences [30]. improve fire safety measures. Acknowledgements Islamia, New Delhi, India. via., virtual, mathematical, or numerical modeling and studies would improve the durability as well as safety of the area. Simulation of possible fire incidences or previous fire case-studies has been known to improve the overall understanding of the incidents and pre-planning strategies [19–21]. Development of such post evacuation strategies would minimize the materialistic loss as well as can zero the Other than the planning of construction and infrastructure skeleton, consideration of the use of specific materials in and around any place is also need to be checked [22]. Utilization of flammable materials such as plastics, oil based paints, use of cotton or storage of highly volatile chemicals seem to be a cause in many instances. Alternatively, non-flammable paints, coatings, fabrics, and construction materials like nanocomposite materials should be used [23–25]. Research on flame retardant materials and nanocomposites are also considered to be one of the alternatives against fire protection and evacuation management systems. Like coating over wooden furniture and cotton fabrics would render them less prone to fire and thus, can act as barrier during incidence of fire [26, 27]. Superhydrophobic and flame retardant tiles in kitchen and heating areas would result in reduction in possible accidents [28, 29]. Further, designing of fire-fighting robots and other engineered bots are required to be brought in lime light for protection and preven- Finally, the strategies which can assist in improving pre, on-going, and post-fire incident management system are more pivotal in creating a fail-safe fire protection mechanism. The research encircling different views with building modeling, escape route planning in heritage buildings and mines, construction material used and prevention protocols are critical in safe keeping future generation including wild life as well as plants. The difficulties which are created due to the excessive urbanization and population boom in addition to poor engineering or planning of residence and work places particularly near forest or wood stock areas are to be addressed. Virtual in silico studies and numerical simulation in analyzing as well as recreating previous fire hazardous cases would provide additional benefits in visualization of the mistakes in following then existing protocols and to further Fahmina Zafar acknowledges Department of Science and Technology, New Delhi, India, for the Women Scientist Scheme (WOS) for Research in Basic/Applied Sciences, Rf# SR/WOSA/CS-97/2016 and Department of Chemistry, Jamia Millia #### Introductory Chapter: Fire Prevention Strategies DOI: http://dx.doi.org/10.5772/intechopen.94037 Fire Safety and Management Awareness 4 2. Comprehensive views Basics steps involved in fire protection of any construction. Three broad categories of the fire protection activities. Figure 2. Figure 1. Although numerous prevention and fire safety manuals have been developed but with modernization redefining fire prevention protocols with time is mandatory [14–16]. However, the process of change is ever dynamic for the inclusion of new information. Incorporation of the output from upcoming research studies for avoiding miss-managements during a fire outburst and remodeling the evacuation strategies are important. The architectural improvements which can further assist the evacuation process and ease the smoke exhaustion process should always be considered. The new engineering strategies based on recent active research should be adopted in the construction of housing societies, particularly in highly populated areas [17, 18]. The implementation of fire safety protocol in pre-build structures via., virtual, mathematical, or numerical modeling and studies would improve the durability as well as safety of the area. Simulation of possible fire incidences or previous fire case-studies has been known to improve the overall understanding of the incidents and pre-planning strategies [19–21]. Development of such post evacuation strategies would minimize the materialistic loss as well as can zero the causalities under such events. Other than the planning of construction and infrastructure skeleton, consideration of the use of specific materials in and around any place is also need to be checked [22]. Utilization of flammable materials such as plastics, oil based paints, use of cotton or storage of highly volatile chemicals seem to be a cause in many instances. Alternatively, non-flammable paints, coatings, fabrics, and construction materials like nanocomposite materials should be used [23–25]. Research on flame retardant materials and nanocomposites are also considered to be one of the alternatives against fire protection and evacuation management systems. Like coating over wooden furniture and cotton fabrics would render them less prone to fire and thus, can act as barrier during incidence of fire [26, 27]. Superhydrophobic and flame retardant tiles in kitchen and heating areas would result in reduction in possible accidents [28, 29]. Further, designing of fire-fighting robots and other engineered bots are required to be brought in lime light for protection and prevention against tragedic incidences [30]. Finally, the strategies which can assist in improving pre, on-going, and post-fire incident management system are more pivotal in creating a fail-safe fire protection mechanism. The research encircling different views with building modeling, escape route planning in heritage buildings and mines, construction material used and prevention protocols are critical in safe keeping future generation including wild life as well as plants. The difficulties which are created due to the excessive urbanization and population boom in addition to poor engineering or planning of residence and work places particularly near forest or wood stock areas are to be addressed. Virtual in silico studies and numerical simulation in analyzing as well as recreating previous fire hazardous cases would provide additional benefits in visualization of the mistakes in following then existing protocols and to further improve fire safety measures. #### Acknowledgements Fahmina Zafar acknowledges Department of Science and Technology, New Delhi, India, for the Women Scientist Scheme (WOS) for Research in Basic/Applied Sciences, Rf# SR/WOSA/CS-97/2016 and Department of Chemistry, Jamia Millia Islamia, New Delhi, India. Fire Safety and Management Awareness ## Author details Anujit Ghosal1,2\* and Fahmina Zafar2 1 School of Lifesciences, Beijing Institute of Sciences, Beijing, PRC 2 Department of Chemistry, Jamia Millia Islamia, New Delhi, India \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 7* Introductory Chapter: Fire Prevention Strategies DOI: http://dx.doi.org/10.5772/intechopen.94037 > [10] M. McNamee, B. Meacham, P. van Hees, L. Bisby, W. Chow, A. Coppalle, R. Dobashi, B. Dlugogorski, R. Fahy, C. Fleischmann, IAFSS agenda 2030 for a fire safe world, Fire Safety Journal, 110 [11] M. de Torres Curth, L. Ghermandi, V. Zimmerman, Living in a world of fire: the population dynamics of Mulinum spinosum in Northwestern Patagonia grasslands, Plant Ecology, (2020) 1-12. [12] A.A. Sheeba, R. Jayaparvathy, Performance modeling of an intelligent emergency evacuation system in buildings on accidental fire occurrence, Safety science, 112 (2019) 196-205. [13] A. Chakraborty, R. Devnath, Fire crisis Management-cesc's experience as Distribution Utility, Water and Energy [14] T. Deave, A. Hawkins, A. Kumar, M. Hayes, N. Cooper, M. Watson, J. Ablewhite, C. Coupland, A. Sutton, G. Majsak-Newman, Evaluating implementation of a fire-prevention injury prevention briefing in children's centres: Cluster randomised controlled trial, PLoS One, 12 (2017) e0172584. [15] M.-Y. Cheng, K.-C. Chiu, Y.-M. Hsieh, I.-T. Yang, J.-S. Chou, Y.-W. Wu, BIM integrated smart monitoring technique for building fire prevention and disaster relief, Automation in Construction, 84 (2017) 14-30. [16] Littlewood J. Smart Fire Performance-Assessment of Occupant Safety in Specialised Dwellings, in: International Conference on Sustainability in Energy and Buildings. Cham: Springer; 2018. pp. 435-444 [17] W. Węgrzyński, P. Sulik, The philosophy of fire safety engineering in the shaping of civil engineering development, Bulletin of the polish International, 62 (2020) 15-21. (2019) 102889. [1] M.J. van Marle, R.D. Field, G.R. van der Werf, I.A.E. de Wagt, R.A. Houghton, L.V. Rizzo, P. Artaxo, K. Tsigaridis, Fire and deforestation dynamics in Amazonia (1973-2014), Global biogeochemical cycles, 31 (2017) [2] A. Patrão, Human and Social Dimensions of Wildland Fire Management and Forest Protection. Restoration, 21 (2020) 73. University, 2020. (2017) 190-204. (2016) 781-785. biotechnology, (2018). [4] W. Zeńczak, A. Krystosik-Gromadzińska, Improvements to a fire safety management system, Polish Maritime Research, 26 (2019) 117-123. [5] H.M. Dijmarescu, Prometheus's Blind Spot: Invoking Rules and Political Histories of Fire, in, Northwestern Engineering. London: IntechOpen; 2019 [8] C. Chen, G. Reniers, Risk assessment of processes and products in industrial [9] W. Knorr, A. Arneth, L. Jiang, Demographic controls of future global fire risk, Nature Climate Change, 6 [7] Y. Khalil, A probabilistic visualflowcharting-based model for consequence assessment of fire and explosion events involving leaks of flammable gases, Journal of Loss Prevention in the Process Industries, 50 [6] Aspragathos N, Dogkas E, Konstantinidis P, Koutmos P, Lamprinou N, Moulianitis VC, et al. From pillars to AI technology-based Forest fire protection systems. In: Artificial Intelligence-Applications in Agriculture and Bio-System [3] T. McDonald, Drought, fire, flood and COVID–complex systems and disruption, Ecological Management & 24-38. References Introductory Chapter: Fire Prevention Strategies DOI: http://dx.doi.org/10.5772/intechopen.94037 #### References Fire Safety and Management Awareness 6 Author details Anujit Ghosal1,2\* and Fahmina Zafar2 provided the original work is properly cited. 1 School of Lifesciences, Beijing Institute of Sciences, Beijing, PRC 2 Department of Chemistry, Jamia Millia Islamia, New Delhi, India © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, \Address all correspondence to: [email protected] [1] M.J. van Marle, R.D. Field, G.R. van der Werf, I.A.E. de Wagt, R.A. Houghton, L.V. Rizzo, P. Artaxo, K. Tsigaridis, Fire and deforestation dynamics in Amazonia (1973-2014), Global biogeochemical cycles, 31 (2017) 24-38. [2] A. Patrão, Human and Social Dimensions of Wildland Fire Management and Forest Protection. [3] T. McDonald, Drought, fire, flood and COVID–complex systems and disruption, Ecological Management & Restoration, 21 (2020) 73. [4] W. Zeńczak, A. Krystosik-Gromadzińska, Improvements to a fire safety management system, Polish Maritime Research, 26 (2019) 117-123. [5] H.M. Dijmarescu, Prometheus's Blind Spot: Invoking Rules and Political Histories of Fire, in, Northwestern University, 2020. [6] Aspragathos N, Dogkas E, Konstantinidis P, Koutmos P, Lamprinou N, Moulianitis VC, et al. From pillars to AI technology-based Forest fire protection systems. In: Artificial Intelligence-Applications in Agriculture and Bio-System Engineering. London: IntechOpen; 2019 [7] Y. Khalil, A probabilistic visualflowcharting-based model for consequence assessment of fire and explosion events involving leaks of flammable gases, Journal of Loss Prevention in the Process Industries, 50 (2017) 190-204. [8] C. Chen, G. Reniers, Risk assessment of processes and products in industrial biotechnology, (2018). [9] W. Knorr, A. Arneth, L. Jiang, Demographic controls of future global fire risk, Nature Climate Change, 6 (2016) 781-785. [10] M. McNamee, B. Meacham, P. van Hees, L. Bisby, W. Chow, A. Coppalle, R. Dobashi, B. Dlugogorski, R. Fahy, C. Fleischmann, IAFSS agenda 2030 for a fire safe world, Fire Safety Journal, 110 (2019) 102889. [11] M. de Torres Curth, L. Ghermandi, V. Zimmerman, Living in a world of fire: the population dynamics of Mulinum spinosum in Northwestern Patagonia grasslands, Plant Ecology, (2020) 1-12. [12] A.A. Sheeba, R. Jayaparvathy, Performance modeling of an intelligent emergency evacuation system in buildings on accidental fire occurrence, Safety science, 112 (2019) 196-205. [13] A. Chakraborty, R. Devnath, Fire crisis Management-cesc's experience as Distribution Utility, Water and Energy International, 62 (2020) 15-21. [14] T. Deave, A. Hawkins, A. Kumar, M. Hayes, N. Cooper, M. Watson, J. Ablewhite, C. Coupland, A. Sutton, G. Majsak-Newman, Evaluating implementation of a fire-prevention injury prevention briefing in children's centres: Cluster randomised controlled trial, PLoS One, 12 (2017) e0172584. [15] M.-Y. Cheng, K.-C. Chiu, Y.-M. Hsieh, I.-T. Yang, J.-S. Chou, Y.-W. Wu, BIM integrated smart monitoring technique for building fire prevention and disaster relief, Automation in Construction, 84 (2017) 14-30. [16] Littlewood J. Smart Fire Performance-Assessment of Occupant Safety in Specialised Dwellings, in: International Conference on Sustainability in Energy and Buildings. Cham: Springer; 2018. pp. 435-444 [17] W. Węgrzyński, P. Sulik, The philosophy of fire safety engineering in the shaping of civil engineering development, Bulletin of the polish academy of sciences. Technical sciences, 64 (2016). [18] E.K. Zavadskas, J. Antucheviciene, T. Vilutiene, H. Adeli, Sustainable decision-making in civil engineering, construction and building technology, Sustainability, 10 (2018) 14. [19] D. Sahu, S. Kumar, S. Jain, A. Gupta, Experimental and numerical simulation studies on diesel pool fire, Fire and Materials, 40 (2016) 1016-1035. [20] B. Lattimer, J. Hodges, A. Lattimer, Using machine learning in physicsbased simulation of fire, Fire Safety Journal, (2020) 102991. [21] P. Ghasemi, A. Babaeinesami, Simulation of fire stations resources considering the downtime of machines: A case study, Journal of Industrial Engineering and Management Studies, 7 (2020) 161-176. [22] E.J. Sugeng, M. de Cock, P.E. Leonards, M. van de Bor, Electronics, interior decoration and cleaning patterns affect flame retardant levels in the dust from Dutch residences, Science of The Total Environment, 645 (2018) 1144-1152. [23] K. Shikinaka, M. Nakamura, R.R. Navarro, Y. Otsuka, Non-flammable and moisture-permeable UV protection films only from plant polymers and clay minerals, Green Chemistry, 21 (2019) 498-502. [24] W. Guo, X. Wang, J. Huang, Y. Zhou, W. Cai, J. Wang, L. Song, Y. Hu, Construction of durable flameretardant and robust superhydrophobic coatings on cotton fabrics for water-oil separation application, Chemical Engineering Journal, (2020) 125661. [25] R. Olawoyin, Nanotechnology: The future of fire safety, Safety science, 110 (2018) 214-221. [26] T. Ma, L. Li, Q. Wang, C. Guo, Construction of intumescent flame retardant and hydrophobic coating on wood substrates based on thiol-ene click chemistry without photoinitiators, Composites Part B: Engineering, 177 (2019) 107357. [27] D. Lin, X. Zeng, H. Li, X. Lai, T. Wu, One-pot fabrication of superhydrophobic and flame-retardant coatings on cotton fabrics via sol-gel reaction, Journal of colloid and interface science, 533 (2019) 198-206. [28] S. Wi, S. Yang, U. Berardi, S. Kim, Assessment of recycled ceramicbased inorganic insulation for improving energy efficiency and flame retardancy of buildings, Environment international, 130 (2019) 104900. [29] F. Zafar, E. Sharmin, Flame Retardants. London: IntechOpen; (2019). DOI: 10.5772/intechopen.82783 Section 2 Effect of Variable Parameters 9* [30] S.S. Priyanka, R. Sangeetha, S. Suvedha, M.G. Vijayalakshmi, Android Controlled Fire Fighting Robot, Ineternational journal of innovative science Engg. and Technology, Volumn, 3 (2017). Section 2	doab	2025-04-07T03:56:59.068315	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 5 }
006df1b2-f518-4d6b-8d96-484202f13086.6	Effect of Variable Parameters Fire Safety and Management Awareness 64 (2016). academy of sciences. Technical sciences, [26] T. Ma, L. Li, Q. Wang, C. Guo, Construction of intumescent flame retardant and hydrophobic coating on wood substrates based on thiol-ene click chemistry without photoinitiators, Composites Part B: Engineering, 177 [27] D. Lin, X. Zeng, H. Li, X. Lai, T. Wu, One-pot fabrication of science, 533 (2019) 198-206. superhydrophobic and flame-retardant coatings on cotton fabrics via sol-gel reaction, Journal of colloid and interface [28] S. Wi, S. Yang, U. Berardi, S. Kim, Assessment of recycled ceramicbased inorganic insulation for improving energy efficiency and flame retardancy of buildings, Environment international, 130 (2019) 104900. [29] F. Zafar, E. Sharmin, Flame Retardants. London: IntechOpen; (2019). DOI: 10.5772/intechopen.82783 [30] S.S. Priyanka, R. Sangeetha, S. Suvedha, M.G. Vijayalakshmi, Android Controlled Fire Fighting Robot, Ineternational journal of innovative science Engg. and Technology, Volumn, 3 (2017). (2019) 107357. [18] E.K. Zavadskas, J. Antucheviciene, T. Vilutiene, H. Adeli, Sustainable decision-making in civil engineering, construction and building technology, [19] D. Sahu, S. Kumar, S. Jain, A. Gupta, Experimental and numerical simulation studies on diesel pool fire, Fire and Materials, 40 (2016) 1016-1035. [20] B. Lattimer, J. Hodges, A. Lattimer, Using machine learning in physicsbased simulation of fire, Fire Safety [21] P. Ghasemi, A. Babaeinesami, Simulation of fire stations resources considering the downtime of machines: A case study, Journal of Industrial Engineering and Management Studies, 7 [22] E.J. Sugeng, M. de Cock, P.E. Leonards, M. van de Bor, Electronics, interior decoration and cleaning patterns affect flame retardant levels in the dust from Dutch residences, Science of The Total Environment, 645 (2018) [23] K. Shikinaka, M. Nakamura, R.R. Navarro, Y. Otsuka, Non-flammable and moisture-permeable UV protection films only from plant polymers and clay minerals, Green Chemistry, 21 (2019) [24] W. Guo, X. Wang, J. Huang, Y. Zhou, W. Cai, J. Wang, L. Song, Y. Hu, Construction of durable flameretardant and robust superhydrophobic coatings on cotton fabrics for water-oil separation application, Chemical Engineering Journal, (2020) 125661. [25] R. Olawoyin, Nanotechnology: The future of fire safety, Safety science, 110 Sustainability, 10 (2018) 14. Journal, (2020) 102991. (2020) 161-176. 1144-1152. 498-502. 8 (2018) 214-221. Chapter 2 Abstract energy. 11 1. Introduction Numerical Study on the Outdoor Wind Effects on Movement Brady Manescau, Khaled Chetehouna, Quentin Serra, In this chapter, a numerical investigation is presented in order to highlight the effects of outdoor wind on smoke movements along a corridor in a compartment. For this, the Computational Fluid Dynamics (CFD) code, fire dynamics simulator (FDS), was used to model the reactive flows in interaction with outdoor wind. The wind velocity is taken between 0 and 12.12 m/s, based on the experimental result data come from the work of Li et al. was performed. From numerical data, it was found that smoke stratification state in the corridor depends on Froude number (Fr) and it can be divided into three cases: stable buoyant stratification (Fr < 0.38), unstable buoyant stratification (0.38 ≤ Fr < 0.76), and failed stratification (Fr ≥ 0.76). When Fr ≥ 0.76, smoke stratification is completely disturbed and smoke occupies the entire volume of the compartment, highlighting a risk of toxicity to people. Indeed, it was observed that the velocity of the outdoor wind influences strongly the concentration of O2, CO2, CO, and visibility in the corridor and smoke exhaust. Moreover, for the input data used in the numerical modelling, the global sensitivity analysis demonstrated that the main parameters affecting the smoke temperature near the ceiling are the mass flux of fuel and the activation Keywords: outdoor wind, CFD, FDS, sensitivity analysis, corridor, smoke spread Since the end of the Second World War, the construction of buildings has experienced an increase in growth due to the increase in the world population and economic growth in recent decades. With many buildings, the problem of housing for people no longer arises. However, by making an inventory of the generally very high number of victims in building fires, these developments present numerous challenges for fire safety engineering. Indeed, for the past 75 years, there have been many fires in large buildings. There is for example, during April 15, 2019, the violent fire that started in the roof of the Notre-Dame de Paris cathedral, and it ravaged the roof and the frame by destroying the roof base and damaging the vault. In order to reduce the number of deaths and property damage, fire safety engineering has focused on understanding the different phenomena present in a building fire [1]. Among these phenomena, Paul et al. [2] and Hull et al. [3] showed that Smoke along a Corridor Aijuan Wang and Eric Florentin #### Chapter 2 ## Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor Brady Manescau, Khaled Chetehouna, Quentin Serra, Aijuan Wang and Eric Florentin #### Abstract In this chapter, a numerical investigation is presented in order to highlight the effects of outdoor wind on smoke movements along a corridor in a compartment. For this, the Computational Fluid Dynamics (CFD) code, fire dynamics simulator (FDS), was used to model the reactive flows in interaction with outdoor wind. The wind velocity is taken between 0 and 12.12 m/s, based on the experimental result data come from the work of Li et al. was performed. From numerical data, it was found that smoke stratification state in the corridor depends on Froude number (Fr) and it can be divided into three cases: stable buoyant stratification (Fr < 0.38), unstable buoyant stratification (0.38 ≤ Fr < 0.76), and failed stratification (Fr ≥ 0.76). When Fr ≥ 0.76, smoke stratification is completely disturbed and smoke occupies the entire volume of the compartment, highlighting a risk of toxicity to people. Indeed, it was observed that the velocity of the outdoor wind influences strongly the concentration of O2, CO2, CO, and visibility in the corridor and smoke exhaust. Moreover, for the input data used in the numerical modelling, the global sensitivity analysis demonstrated that the main parameters affecting the smoke temperature near the ceiling are the mass flux of fuel and the activation energy. Keywords: outdoor wind, CFD, FDS, sensitivity analysis, corridor, smoke spread #### 1. Introduction Since the end of the Second World War, the construction of buildings has experienced an increase in growth due to the increase in the world population and economic growth in recent decades. With many buildings, the problem of housing for people no longer arises. However, by making an inventory of the generally very high number of victims in building fires, these developments present numerous challenges for fire safety engineering. Indeed, for the past 75 years, there have been many fires in large buildings. There is for example, during April 15, 2019, the violent fire that started in the roof of the Notre-Dame de Paris cathedral, and it ravaged the roof and the frame by destroying the roof base and damaging the vault. In order to reduce the number of deaths and property damage, fire safety engineering has focused on understanding the different phenomena present in a building fire [1]. Among these phenomena, Paul et al. [2] and Hull et al. [3] showed that smoke is the main cause of death due to toxicity. Indeed, smoke plume can be hazardous for people in two different ways: the toxic gases in smoke, such as carbon dioxide, are a fatal hazard [2, 3], and the smoke can make it difficult to rescue and evacuate people as it reduces visibility. In a compartment fire, it is therefore very important to know the characteristics of the smoke spread. The parameters that influence the smoke spread are mechanical ventilation and external atmospheric conditions. Generally, mechanical ventilation ensures smoke exhaust; however, external atmospheric conditions can disturb smoke flow. Moreover, smoke flows depend essentially on physical properties such as expansion, thermal pressure, thermal buoyancy, and wind effect. Variation in one of these parameters, such as the wind speed, can affect strongly smoke behavior [4, 5]. Considering this possibility, it is important to highlight the effects of external atmospheric conditions on smoke spread in a compartment. Most of the numerical simulations that focused on the propagation of smoke in a ventilated or unventilated enclosure studied the level of smoke stratification as a function of the temperature profile and velocity. However, these numerical studies do not consider the effect of the external wind on smoke stratification in a corridor adjacent to a burning room with an opening. Using the data obtained by Li et al. [8], the aim of the present study was to highlight the ability of the fire dynamics simulator (FDS) to study smoke behavior according to the variation in outdoor wind velocity. This work, through a mesh resolution [15, 16], consists in reproducing the experimental conditions obtained in the work of Li et al. [8]. Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor In this chapter, a CFD approach was proposed to evaluate the effects of outdoor wind on the smoke spread induced by an adjacent compartment fire. In order to highlight the influence of the input parameters used as initial conditions in the computational modelling, a global sensitivity analysis was performed. For this, Section 2 presents an overview of the global sensitivity analysis methodology with polynomial chaos expansion. Section 3 defines the physical and numerical modelling, Section 4 focuses on numerical results and global sensitivity analysis, and the The aim of sensitivity analysis is to quantify the influence of the variation of an input parameter on the variation of an output, also called quantity of interest. In the present study, the quantity of interest is the predicted smoke temperature near the ceiling (X = 5.4 m, Y = 0.5 m, Z = 85 cm), filtered by a Savitzky-Golay algorithm (third-order) to eliminate high-frequency variations of temperature. It is expressed as a mapping of the input parameters xi for i ¼ 1 … r, where r is the number of parameters, and the dependence in time is omitted to simplify the notations as: Generally, there are two kinds of sensitivity analysis: local sensitivity analysis and global sensitivity analysis. The local sensitivity analysis is a simple approach in which the sensitivity indices are directly related to the derivatives of the quantity of interest with respect to each parameter [17–19]. It is called local because the local indices are only valid in a neighborhood of the nominal value [20]. While local approaches are restricted to the vicinity of the prescribed deterministic values, global sensitivity takes To extend the approach in the case of larger variations of parameters, a probabilistic framework is adopted. Lacking knowledge on the probability density functions of the inputs, we assume that each of the parameters follows a uniform law Of interest in this chapter are the Sobol sensitivity indices. These indices are often associated to an analysis of variance (ANOVA) decomposition, which consists in the decomposition of the model response into main effects and > Xr 1≤i<j≤r Mij xi, xj � � <sup>þ</sup> … <sup>þ</sup> M1, … ,rð Þ x1, … , xr (2) into account the entire domain of variation of each parameter. i¼1 Mið Þþ xi with a �10% variation around its nominal value. interactions [21]: 13 <sup>T</sup> ¼ Mð Þ¼ x1, … , xr M0 <sup>þ</sup>X<sup>r</sup> T ¼ Mð Þ x1, … , xr (1) conclusion is presented in Section 5. DOI: http://dx.doi.org/10.5772/intechopen.92978 2.1 Global sensitivity analysis 2. Sensitivity analysis methodology Indeed, during a fire in a room, the presence of external wind through an opening contributes to disturbance of the smoke flow, which can impair the extraction process, thereby increasing the risk of death. In this context, many studies have been carried out in recent years to provide knowledge for fire safety engineering, including many full-scale [6, 7] and reduced scale [8, 9] experimental investigations. At full scale, Tian et al. [6] showed that the more the wind velocity increases, the more the smoke temperature near the floor increases, in order to converge to the smoke temperature near the ceiling. Considering this, they highlighted that above a certain critical value of outdoor wind velocity, smoke stratification was disturbed, and smoke occupied the entire volume of the compartment. From scaling laws [10], small-scale experimental tests have been developed. Indeed, Li et al. [8] studied the influence of external wind velocity on the smoke flow in a small-scale facility. They showed that the driving forces of smoke flow through a high-rise building were modified according to the intensity of the external wind. In addition to the experimental studies cited above, numerical simulations on smoke propagation in a compartment have also been conducted. For example, Li et al. [11] simulated smoke flows in a reduced scale (1:12) corridor under natural ventilation conditions using the CFD code FDS. In their work, they compared numerical data with experimental data and highlighted that FDS was able to simulate the temperature field and the level of smoke stratification for different heat release rates (HRR). In another example, Weng et al. [12] performed numerical simulations on the smoke flow in a subway tunnel fire equipped with an extraction system. Their results revealed that the temperature and the level of smoke stratification under the tunnel ceiling in the longitudinal direction increased with the HRR. Considering the numerical studies presented above, it is shown that, using nice initial and boundary conditions, it is possible to make accurate simulations. Thus, in order to obtain accurate output results, it is necessary to define the input data correctly by carrying out a sensitivity analysis in order to find out the input parameters having the most influence on the output data. Two kinds of approach are classically used to achieve this: local and global approaches. For example, Batiot et al. [13] applied local and global sensitivity analysis on Arrhenius parameters in order to describe the kinetics of solid thermal degradation during fire phenomena, by determining four parameters (A, E, n, and ν). They stressed the specific role of A and E on the equation and showed the role and the influence of these parameters in the differential equation used to model the mass loss rate of a solid fuel as a function of the temperature and time. In a second example, Xiao et al. [14] applied global sensitivity analysis to an environmental model named Level E. The sensitivity indices used the energy distribution of the model output over different frequency bands as the quantitative feature of the model output. Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 Most of the numerical simulations that focused on the propagation of smoke in a ventilated or unventilated enclosure studied the level of smoke stratification as a function of the temperature profile and velocity. However, these numerical studies do not consider the effect of the external wind on smoke stratification in a corridor adjacent to a burning room with an opening. Using the data obtained by Li et al. [8], the aim of the present study was to highlight the ability of the fire dynamics simulator (FDS) to study smoke behavior according to the variation in outdoor wind velocity. This work, through a mesh resolution [15, 16], consists in reproducing the experimental conditions obtained in the work of Li et al. [8]. In this chapter, a CFD approach was proposed to evaluate the effects of outdoor wind on the smoke spread induced by an adjacent compartment fire. In order to highlight the influence of the input parameters used as initial conditions in the computational modelling, a global sensitivity analysis was performed. For this, Section 2 presents an overview of the global sensitivity analysis methodology with polynomial chaos expansion. Section 3 defines the physical and numerical modelling, Section 4 focuses on numerical results and global sensitivity analysis, and the conclusion is presented in Section 5. #### 2. Sensitivity analysis methodology #### 2.1 Global sensitivity analysis smoke is the main cause of death due to toxicity. Indeed, smoke plume can be hazardous for people in two different ways: the toxic gases in smoke, such as carbon dioxide, are a fatal hazard [2, 3], and the smoke can make it difficult to rescue and evacuate people as it reduces visibility. In a compartment fire, it is therefore very important to know the characteristics of the smoke spread. The parameters that influence the smoke spread are mechanical ventilation and external atmospheric conditions. Generally, mechanical ventilation ensures smoke exhaust; however, external atmospheric conditions can disturb smoke flow. Moreover, smoke flows depend essentially on physical properties such as expansion, thermal pressure, thermal buoyancy, and wind effect. Variation in one of these parameters, such as the wind speed, can affect strongly smoke behavior [4, 5]. Considering this possibility, it is important to highlight the effects of external atmospheric conditions on Indeed, during a fire in a room, the presence of external wind through an opening contributes to disturbance of the smoke flow, which can impair the extraction process, thereby increasing the risk of death. In this context, many studies have been carried out in recent years to provide knowledge for fire safety engineering, including many full-scale [6, 7] and reduced scale [8, 9] experimental At full scale, Tian et al. [6] showed that the more the wind velocity increases, the more the smoke temperature near the floor increases, in order to converge to the smoke temperature near the ceiling. Considering this, they highlighted that above a certain critical value of outdoor wind velocity, smoke stratification was disturbed, and smoke occupied the entire volume of the compartment. From scaling laws [10], small-scale experimental tests have been developed. Indeed, Li et al. [8] studied the influence of external wind velocity on the smoke flow in a small-scale facility. They showed that the driving forces of smoke flow through a high-rise building were In addition to the experimental studies cited above, numerical simulations on smoke propagation in a compartment have also been conducted. For example, Li et al. [11] simulated smoke flows in a reduced scale (1:12) corridor under natural ventilation conditions using the CFD code FDS. In their work, they compared numerical data with experimental data and highlighted that FDS was able to simulate the temperature field and the level of smoke stratification for different heat release rates (HRR). In another example, Weng et al. [12] performed numerical simulations on the smoke flow in a subway tunnel fire equipped with an extraction system. Their results revealed that the temperature and the level of smoke stratification under the tunnel ceiling in the longitudinal direction increased with the HRR. Considering the numerical studies presented above, it is shown that, using nice initial and boundary conditions, it is possible to make accurate simulations. Thus, in order to obtain accurate output results, it is necessary to define the input data correctly by carrying out a sensitivity analysis in order to find out the input parameters having the most influence on the output data. Two kinds of approach are classically used to achieve this: local and global approaches. For example, Batiot et al. [13] applied local and global sensitivity analysis on Arrhenius parameters in order to describe the kinetics of solid thermal degradation during fire phenomena, by determining four parameters (A, E, n, and ν). They stressed the specific role of A and E on the equation and showed the role and the influence of these parameters in the differential equation used to model the mass loss rate of a solid fuel as a function of the temperature and time. In a second example, Xiao et al. [14] applied global sensitivity analysis to an environmental model named Level E. The sensitivity indices used the energy distribution of the model output over different frequency modified according to the intensity of the external wind. bands as the quantitative feature of the model output. smoke spread in a compartment. Fire Safety and Management Awareness investigations. 12 The aim of sensitivity analysis is to quantify the influence of the variation of an input parameter on the variation of an output, also called quantity of interest. In the present study, the quantity of interest is the predicted smoke temperature near the ceiling (X = 5.4 m, Y = 0.5 m, Z = 85 cm), filtered by a Savitzky-Golay algorithm (third-order) to eliminate high-frequency variations of temperature. It is expressed as a mapping of the input parameters xi for i ¼ 1 … r, where r is the number of parameters, and the dependence in time is omitted to simplify the notations as: $$\mathbf{T} = \mathcal{M}(\mathbf{x\_1}, \dots, \mathbf{x\_r}) \tag{1}$$ Generally, there are two kinds of sensitivity analysis: local sensitivity analysis and global sensitivity analysis. The local sensitivity analysis is a simple approach in which the sensitivity indices are directly related to the derivatives of the quantity of interest with respect to each parameter [17–19]. It is called local because the local indices are only valid in a neighborhood of the nominal value [20]. While local approaches are restricted to the vicinity of the prescribed deterministic values, global sensitivity takes into account the entire domain of variation of each parameter. To extend the approach in the case of larger variations of parameters, a probabilistic framework is adopted. Lacking knowledge on the probability density functions of the inputs, we assume that each of the parameters follows a uniform law with a �10% variation around its nominal value. Of interest in this chapter are the Sobol sensitivity indices. These indices are often associated to an analysis of variance (ANOVA) decomposition, which consists in the decomposition of the model response into main effects and interactions [21]: $$\mathbf{T} = \mathcal{M}(\mathbf{x\_1}, \dots, \mathbf{x\_r}) = \mathbf{M}\_0 + \sum\_{i=1}^r \mathbf{M}\_i(\mathbf{x\_i}) + \sum\_{1 \le i < j \le r}^r \mathbf{M}\_{\overline{\mathbf{z}}}(\mathbf{x\_i}, \mathbf{x\_j}) + \dots + \mathbf{M}\_{1, \dots, r}(\mathbf{x\_1}, \dots, \mathbf{x\_r}) \tag{2}$$ The decomposition is unique if summands satisfy the properties [20]: $$\mathbf{M}\_0 = \int \mathcal{M}(\mathbf{x}\_1, \dots, \mathbf{x}\_r) d\mathbf{x}\_1 \dots d\mathbf{x}\_r \tag{3}$$ indices ~ ~ Si ¼ P α∈ Af g<sup>i</sup> α Y2 α <sup>D</sup><sup>~</sup> with Af g<sup>i</sup> ~ Sti ¼ Si, and the total sensitivity indices ~ DOI: http://dx.doi.org/10.5772/intechopen.92978 <sup>M</sup><sup>~</sup> <sup>0</sup> <sup>¼</sup> ð <sup>D</sup><sup>~</sup> <sup>¼</sup> ð P α ∈ Atf g<sup>i</sup> α Y2 α <sup>D</sup><sup>~</sup> with Atf g<sup>i</sup> projection or by regression [23]. f g <sup>Y</sup><sup>α</sup> <sup>¼</sup> argmin <sup>1</sup> 70 samples. 15 3. Numerical modelling 3.1 Governing equations the metamodel. This regression approach leads to: Q X Q @ q¼1 analytical functions of the chaos coefficients Y<sup>α</sup> [20]: <sup>M</sup><sup>~</sup> ð Þ� x1, … , xr <sup>M</sup><sup>~</sup> <sup>0</sup> � �<sup>2</sup> Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor <sup>α</sup> ¼ αð Þ<sup>1</sup> , … , αð Þ<sup>r</sup> <sup>α</sup> ¼ αð Þ<sup>1</sup> , … , αð Þ<sup>r</sup> To compute the chaos coefficients Yα, intrusive and nonintrusive approaches can be used. The intrusive approach [22] consists in using PC expansion as an a priori function in the numerical solver. The development of a specific code is needed. It results in a single run of a very large problem. Here, we only consider nonintrusive techniques, in which the chaos coefficients are evaluated with repeated runs of a determinist program. Chaos coefficients can be evaluated by Here, we apply the second technique: it consists in searching the set of coefficients minimizing in the least-squares sense, the L2 distance between the model and α∈ A<sup>α</sup> <sup>Y</sup>αΦα x1,q, … , xr,q � � !<sup>2</sup> <sup>0</sup> The system is solved in a mean least-squares sense with a number Q of sampling points x1,q, … , xr,q � � larger than the number of coefficients to be identified. Typically, in the literature, the number of sampling points is equal to twice the number of polynomial coefficients. In this study, the metamodel is computed using secondorder Legendre polynomials. This leads to Np ¼ 36 stochastic modes, so that the number of sampling points is Q ¼ 72. Roots of the third-order Legendre polynomial, <sup>Ψ</sup><sup>3</sup> xi,q � � <sup>¼</sup> 0, are chosen as sampling points. The results presented here were validated using the jackknife technique with 100 replications of a random subset of The simulations were carried out using the CFD code fire dynamics simulator (FDS) version 6.5.3 [24]. It solves the Navier-Stokes equations using an explicit finite difference scheme. As a CFD code, FDS models the thermally driven flow with an emphasis on smoke and heat transport. It is a large eddy simulation (LES) model using a uniform mesh and has parallel computing capability using messagepassing interface (MPI) [25]. Reactive flows are modelled using a turbulence model based on a LES approach, a combustion model based on the eddy dissipation <sup>M</sup> x1,q, … , xr,q � � � <sup>X</sup> Sti of the metamodel can be computed as α∈ Aαnf g 0, … , 0 � � such as : <sup>α</sup>ð Þ<sup>i</sup> 6¼ <sup>0</sup> � � (12) Y2 <sup>α</sup> (10) 1 A (13) (11) M x <sup>~</sup> ð Þ 1, … , xr dx1 … dxr <sup>¼</sup> <sup>Y</sup>f g 0, … ,0 (9) � � such as : <sup>α</sup>ð Þ<sup>i</sup> 6¼ 0 and <sup>α</sup>ð Þ<sup>j</sup> 6¼ 0 for j 6¼ <sup>i</sup> � � dx1 … dxr <sup>¼</sup> <sup>X</sup> $$\int \mathcal{M}(\mathbf{x}\_{\mathbf{i}\_1}, \dots, \mathbf{x}\_{\mathbf{i}\_s}) \, d\mathbf{x}\_{\mathbf{i}\_1} \dots d\mathbf{x}\_{\mathbf{i}\_s} = \mathbf{0} \text{ for } \mathbf{1} \le \mathbf{i}\_1 < \dots < \mathbf{i}\_s \le \mathbf{r} \tag{4}$$ The variance of the model response according to variation of inputs can be derived as a sum of partial variances as follows: $$\text{var}(\mathbf{T}) = \mathbf{D} = \sum\_{i=1}^{\mathbf{r}} \mathbf{D}\_{\mathbf{i}} + \sum\_{1 \le i < j \le r}^{\mathbf{r}} \mathbf{D}\_{\mathbf{i}\<\mathbf{j}} + \dots + \mathbf{D}\_{1,\dots,\mathbf{r}} \tag{5}$$ The partial variances Di1, … ,is are defined by: $$\mathbf{D}\_{\mathbf{i}\_{1},\ldots,\mathbf{i}\_{\star}} = \int \mathbf{M}\_{\mathbf{i}\_{1},\ldots,\mathbf{i}\_{\star}}(\mathbf{x}\_{\mathbf{i}\_{1}},\ldots,\mathbf{x}\_{\mathbf{i}\_{\star}})^{2} \mathbf{dx}\_{\mathbf{i}\_{1}}\ldots\mathbf{dx}\_{\mathbf{i}\_{\star}} \tag{6}$$ Then, the Sobol indices can be derived according to: $$\mathbf{S}\_{\mathbf{i}\_1,\dots,\mathbf{i}\_s} = \frac{\mathbf{D}\_{\mathbf{i}\_1,\dots,\mathbf{i}\_s}}{\mathbf{D}} \tag{7}$$ Crude Monte Carlo simulations or sampling-based techniques can be applied to obtain these indices, but the associated numerical is prohibitive for computationally demanding models such as those used in this chapter. To overcome this difficulty, the exact model provided by simulations was substituted by an analytical approximation, called metamodel, for which the computation of Sobol indices is exact and analytical. In this chapter, a polynomial chaos expansion was used as metamodel to derive the sensitivity indices. #### 2.2 Polynomial chaos expansion The polynomial chaos (PC) expansion consists in the projection of the model M on the space spanned by a family of Np orthogonal polynomials: $$\mathcal{M}(\mathbf{x}\_1, \dots, \mathbf{x}\_\mathbf{r}) \approx \tilde{\mathcal{M}}(\mathbf{x}\_1, \dots, \mathbf{x}\_\mathbf{r}) = \sum\_{a \in A\_a} \mathbf{Y}\_a \Phi\_a(\mathbf{x}\_1, \dots, \mathbf{x}\_\mathbf{r}) \tag{8}$$ where A<sup>α</sup> is a finite set of vectors of positive integers α ¼ αð Þ<sup>1</sup> , … , αð Þ<sup>r</sup> � � such as cardð Þ¼ A<sup>α</sup> Np. Each of the multivariate polynomials Φα can be expressed as a product of monovariate polynomials Ψαð Þ<sup>i</sup> of order αð Þ<sup>i</sup> : $$\Phi\_a(\mathbf{x\_1}, \dots, \mathbf{x\_r}) = \Psi\_{a\_{(1)}}(\mathbf{x\_1}) \times \dots \times \Psi\_{a\_{(r)}}(\mathbf{x\_r})$$ Legendre polynomials were used here because of the assumption of a uniform probability density function for each input parameter. To reduce the number of stochastic coefficients and thus the computational burden, a classical truncation criterion consists in prescribing the constraint: P<sup>r</sup> <sup>i</sup>¼<sup>1</sup>αð Þ<sup>i</sup> <sup>≤</sup>p, where p is the maximum order allowed for each monovariate polynomial. The interest in such a decomposition is that, due to orthonormal properties of the family of polynomials, the mean M~ 0, the variance D, the first-order Sobol ~ Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 indices ~ Si, and the total sensitivity indices ~ Sti of the metamodel can be computed as analytical functions of the chaos coefficients Y<sup>α</sup> [20]: $$ \tilde{\mathbf{M}}\_0 = \int \tilde{\mathbf{M}}(\mathbf{x}\_1, \dots, \mathbf{x}\_r) d\mathbf{x}\_1 \dots d\mathbf{x}\_r = \mathbf{Y}\_{\{0,\dots,0\}} \tag{9} $$ $$\tilde{\mathbf{D}} = \int \left( \tilde{\mathcal{M}}(\mathbf{x}\_1, \dots, \mathbf{x}\_r) - \tilde{\mathbf{M}}\_0 \right)^2 d\mathbf{x}\_1 \dots d\mathbf{x}\_r = \sum\_{a \in \mathcal{A}\_\mathbf{r} \backslash \{0, \dots, 0\}} \mathbf{Y}\_a^2 \tag{10}$$ $$\tilde{\mathbf{S}}\_{\mathbf{i}} = \frac{\sum\_{a \in \mathcal{A}\_{\mathbf{i}}^{\{\bar{\mathbf{i}}\}}} \mathbf{Y}\_{\alpha}^{2}}{\tilde{\mathbf{D}}} \text{ with } \mathcal{A}\_{\mathbf{a}}^{\{\bar{\mathbf{i}}\}} = \left\{ (\mathfrak{a}\_{(1)}, \dots, \mathfrak{a}\_{(r)}) \text{ such as } : \mathfrak{a}\_{(\bar{\mathbf{i}})} \neq \mathbf{0} \text{ and } \mathfrak{a}\_{(\bar{\mathbf{j}})} \neq \mathbf{0} \text{ for } \mathbf{j} \neq \mathbf{i} \right\} \tag{11}$$ $$\tilde{\mathbf{St}}\_{\mathbf{i}} = \frac{\sum\_{a \in \mathcal{A}\_{\mathbf{u}}^{\{\bar{i}\}}} \mathbf{Y}\_{\mathbf{a}}^{2}}{\tilde{\mathbf{D}}} \text{ with } \mathbf{A}\_{\mathbf{u}}^{\{\bar{i}\}} = \left\{ \left( \mathfrak{a}\_{(1)}, \dots, \mathfrak{a}\_{(r)} \right) \text{ such as } : \mathfrak{a}\_{(\bar{i})} \neq \mathbf{0} \right\} \tag{12}$$ To compute the chaos coefficients Yα, intrusive and nonintrusive approaches can be used. The intrusive approach [22] consists in using PC expansion as an a priori function in the numerical solver. The development of a specific code is needed. It results in a single run of a very large problem. Here, we only consider nonintrusive techniques, in which the chaos coefficients are evaluated with repeated runs of a determinist program. Chaos coefficients can be evaluated by projection or by regression [23]. Here, we apply the second technique: it consists in searching the set of coefficients minimizing in the least-squares sense, the L2 distance between the model and the metamodel. This regression approach leads to: $$\mathbb{V}\left\{\mathbf{Y}\_{a}\right\} = \operatorname\{argmin}\left(\frac{1}{\mathbf{Q}}\sum\_{\mathbf{q}=1}^{\mathbf{Q}}\left(\mathcal{M}\left(\mathbf{x}\_{\text{1,q}},\dots,\mathbf{x}\_{\text{r,q}}\right) - \sum\_{a\in A\_{a}} \mathbf{Y}\_{a}\Phi\_{a}\left(\mathbf{x}\_{\text{1,q}},\dots,\mathbf{x}\_{\text{r,q}}\right)\right)^{2}\right) \tag{13}$$ The system is solved in a mean least-squares sense with a number Q of sampling points x1,q, … , xr,q � � larger than the number of coefficients to be identified. Typically, in the literature, the number of sampling points is equal to twice the number of polynomial coefficients. In this study, the metamodel is computed using secondorder Legendre polynomials. This leads to Np ¼ 36 stochastic modes, so that the number of sampling points is Q ¼ 72. Roots of the third-order Legendre polynomial, <sup>Ψ</sup><sup>3</sup> xi,q � � <sup>¼</sup> 0, are chosen as sampling points. The results presented here were validated using the jackknife technique with 100 replications of a random subset of 70 samples. #### 3. Numerical modelling* #### 3.1 Governing equations The simulations were carried out using the CFD code fire dynamics simulator (FDS) version 6.5.3 [24]. It solves the Navier-Stokes equations using an explicit finite difference scheme. As a CFD code, FDS models the thermally driven flow with an emphasis on smoke and heat transport. It is a large eddy simulation (LES) model using a uniform mesh and has parallel computing capability using messagepassing interface (MPI) [25]. Reactive flows are modelled using a turbulence model based on a LES approach, a combustion model based on the eddy dissipation The decomposition is unique if summands satisfy the properties [20]: The variance of the model response according to variation of inputs can be Di <sup>þ</sup> <sup>X</sup><sup>r</sup> Mi1, … is xi1 , … , xis ð Þ<sup>2</sup> Si1, … ,is <sup>¼</sup> Di1, … ,is Crude Monte Carlo simulations or sampling-based techniques can be applied to obtain these indices, but the associated numerical is prohibitive for computationally demanding models such as those used in this chapter. To overcome this difficulty, the exact model provided by simulations was substituted by an analytical approximation, called metamodel, for which the computation of Sobol indices is exact and analytical. In this chapter, a polynomial chaos expansion was used as metamodel to The polynomial chaos (PC) expansion consists in the projection of the model M where A<sup>α</sup> is a finite set of vectors of positive integers α ¼ αð Þ<sup>1</sup> , … , αð Þ<sup>r</sup> as cardð Þ¼ A<sup>α</sup> Np. Each of the multivariate polynomials Φα can be expressed as a Φαð Þ¼ x1, … , xr Ψαð Þ<sup>1</sup> ð Þ� x1 … � Ψαð Þ<sup>r</sup> ð Þ xr Legendre polynomials were used here because of the assumption of a uniform probability density function for each input parameter. To reduce the number of stochastic coefficients and thus the computational burden, a classical truncation The interest in such a decomposition is that, due to orthonormal properties of the family of polynomials, the mean M~ 0, the variance D, the first-order Sobol X α∈ A<sup>α</sup> 1≤i<j≤r Mð Þ x1, … , xr dx1 … :dxr (3) Dij þ … þ D1, … ,r (5) dxi1 … :dxis (6) YαΦαð Þ x1, … , xr (8) <sup>i</sup>¼<sup>1</sup>αð Þ<sup>i</sup> <sup>≤</sup>p, where p is the ~ � � such <sup>D</sup> (7) M xi1 , … , xis ð Þ dxi1 … :dxis ¼ 0 for 1≤i1 < … <is ≤r (4) M0 ¼ ð derived as a sum of partial variances as follows: var Tð Þ¼ <sup>D</sup> <sup>¼</sup> <sup>X</sup><sup>r</sup> The partial variances Di1, … ,is are defined by: Di1, … ,is ¼ i¼1 ð on the space spanned by a family of Np orthogonal polynomials: Mð Þ x1, … , xr <sup>≈</sup>M<sup>~</sup> ð Þ¼ x1, … , xr product of monovariate polynomials Ψαð Þ<sup>i</sup> of order αð Þ<sup>i</sup> : criterion consists in prescribing the constraint: P<sup>r</sup> 14 maximum order allowed for each monovariate polynomial. Then, the Sobol indices can be derived according to: ð Fire Safety and Management Awareness derive the sensitivity indices. 2.2 Polynomial chaos expansion concept (EDC), and a thermal radiation model based on a gray gas model for the radiation absorption coefficient [15, 16]. The models are based on the numerical solving of Navier-Stokes equations. These equations calculate mass, momentum, species, and energy conservation [25]: $$\frac{\partial \rho}{\partial \mathbf{t}} + \frac{\partial}{\partial \mathbf{x}\_{\circ}} \left(\rho \mathbf{u}\_{\circ}\right) = \mathbf{0} \tag{14}$$ the scaling law of Froude modelling [12, 13]. Varying the wind velocity, eight experiments were conducted at an HRR of 96.2 kW, equaling 1.5 MW at full-scale. The experiments were carried out with ambient temperature ranging from 6 to 16°C. K-type thermocouples with an accuracy of �1°C were used for the temperature measurements in the corridor and fire room. Hot wire wind speed meters were Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor In order to model the geometry and the boundary conditions of the setup used during the experimental tests [8], the walls of the corridor and fireroom were made In simulations, the boundary condition at the window was modeled as an opening in the case without wind. With wind, a constant flow rate was set at the window using the velocity boundary used in the CFD code. In order to remain consistent with the experimental tests, a waiting time of 150 s was defined before activation of the outdoor wind velocity in the modelling. The simulations were performed in eight cases (Vw = 0, 1, 2, 3, 4, 5, 6, and 7 m/s, which correspond to Vw = 0, 1.73, 3.46, 5.20, 6.93, 8.66, 10.39, and 12.12 m/s full-scale). Similarly, the simulation results were converted into full-scale data according to the Froude number. The smoke temperature, smoke velocity, concentration of O2 and CO, and visibility in the corridor were analyzed by setting different devices in the plane (Y = 0.5 m), near the exit of the corridor (X = 5.4 m), at different heights (Z = 85, 70, 55, 40, and 25 cm). Moreover, other observations were carried out about the distribution of temperature, velocity, concentration of O2 and CO2, and visibility thanks to For numerical studies, it is important to choose the correct mesh size in order to obtain accurate simulation results. FDS provides a range of mesh sizes for mesh resolution. From a Poisson solver based on the fast Fourier transform (FFT), it is possible to obtain good numerical resolution by solving the governing equations. The mesh size was chosen in accordance with the recommendations made in the numerical studies [15, 16]. An optimal mesh size should meet two requirements: good results in terms of accuracy and a short calculation time. The optimal mesh nominal mesh size and D\* is the characteristic fire diameter [24]. The characteristic ρ∞cpT<sup>∞</sup> !2 ffiffi g p =5 <sup>D</sup><sup>∗</sup> <sup>¼</sup> <sup>Q</sup>\_ size of the domain is given by the nondimensional expression <sup>D</sup><sup>∗</sup> fire diameter D\* is determined using Eq. (18): , a thermal conductivity of 46 W/(m�K)�<sup>1</sup> = <sup>∂</sup>x, where ∂x is the (18) , a applied to measure the velocity of smoke. Schematic view of the experimental corridor and fire room. DOI: http://dx.doi.org/10.5772/intechopen.92978 Figure 1. of steel having a density of 7850 kg/m<sup>3</sup> slice fields in the plane Y = 0.5 m. 3.4 Mesh size resolution 17 specific heat of 0.5 kJ/(kg�K), and an emissivity of 0.9. $$\frac{\partial}{\partial \mathbf{t}} \left( \rho \mathbf{u}\_{\mathbf{i}} \right) + \frac{\partial}{\partial \mathbf{x}\_{\mathbf{j}}} \left( \rho \mathbf{u}\_{\mathbf{i}} \mathbf{u}\_{\mathbf{j}} \right) = -\frac{\partial \mathbf{p}}{\partial \mathbf{x}\_{\mathbf{i}}} + \rho \mathbf{g}\_{\mathbf{i}} + \frac{\partial}{\partial \mathbf{x}\_{\mathbf{j}}} \left\{ \mu \left( \frac{\partial \mathbf{u}\_{\mathbf{i}}}{\partial \mathbf{x}\_{\mathbf{j}}} + \frac{\partial \mathbf{u}\_{\mathbf{j}}}{\partial \mathbf{x}\_{\mathbf{i}}} - \frac{2}{3} \delta\_{\mathbf{i}\mathbf{j}} \frac{\partial \mathbf{u}\_{\mathbf{i}}}{\partial \mathbf{x}\_{\mathbf{i}}} \right) \right\} \tag{15}$$ $$\frac{\partial \rho \mathbf{Y}\_{\mathbf{k} \ast}}{\partial \mathbf{t}} + \frac{\partial}{\partial \mathbf{x}\_{\mathbf{j}}} \rho \mathbf{u}\_{\mathbf{j}} \mathbf{Y}\_{\mathbf{k} \ast} = \frac{\partial}{\partial \mathbf{x}\_{\mathbf{j}}} \left\{ (\rho \mathbf{D})\_{\mathbf{k} \ast} \frac{\partial}{\partial \mathbf{x}\_{\mathbf{j}}} \mathbf{Y}\_{\mathbf{k} \ast} \right\} + \dot{\mathbf{o}}\_{\mathbf{k} \ast}^{\prime\prime} \tag{16}$$ $$\frac{\partial}{\partial \mathbf{t}}(\rho \mathbf{h}) + \frac{\partial}{\partial \mathbf{x}\_{\circ}}(\rho \mathbf{u}\_{\circ} \mathbf{h}) = \frac{d\mathbf{p}}{d\mathbf{t}} + \dot{\mathbf{q}}''' + \lambda \frac{\partial^2}{\partial \mathbf{x}\_{\circ}^2} \mathbf{T} + \sum \frac{\partial}{\partial \mathbf{x}\_{\circ}} \left\{ \mathbf{h}\_{\mathbf{k}\} (\rho \mathbf{D})\_{\mathbf{k}\} \frac{\partial}{\partial \mathbf{x}\_{\circ}} \mathbf{Y}\_{\mathbf{k}\} \right\} - \frac{\partial}{\partial \mathbf{x}\_{\circ}} \dot{\mathbf{q}}''\_{\mathbf{r}, \circ} \tag{17}$$ where Eq. (14) represents the mass conservation equation, Eq. (15) represents the momentum conservation equation, Eq. (16) represents the species conservation equation, and Eq. (17) represents the energy conservation equation. #### 3.2 Fire modelling* The modelling was carried out using the Deardorff turbulence model and extinction model based on a critical flame temperature. The combustion model is based on the finite rate combustion using Arrhenius parameters (A: pre-exponential factor and Ea: activation energy). The fire source was modelled as a gas burner using butane as fuel with mass flux given by the experimental data [8]. The combustion heat of butane is 45182.83 kJ/kg. #### 3.3 Computational domain and boundary conditions The experimental setup used as reference in the current numerical study represents a reduced scale (1:3) of a facility which contains a corridor and a fire room [8]. As shown in Figure 1, the dimensions of the corridor were 5.5 m (length) � 0.7 m (width) � 0.9 m (height) and the dimensions of the fire room were 2.0 m (length) � 1.7 m (width) � 1.0 m (height). The corridor and fire room were connected by a door whose dimensions were 0.7 m long by 0.3 m wide. The window in the fire room was opposite to the door and its dimensions were 0.5 m (width) � 0.5 m (height). The ceilings and floors of the corridor and fire room were made of steel plate with a thickness of 2.5 mm. As shown in Figure 1, the fire source was in the middle of the fire room, and it was defined as a gas burner using liquefied petroleum gas as fuel. The fuel supply rates of the gas burner were controlled and monitored by a flow meter. The HRR in the experiments was determined by multiplying the mass flow rate and the combustion heat of liquefied petroleum gas. The fire size can be scaled up to 96.2 kW of HRR, which corresponds to 1.5 MW full-scale. The wind can blow into the fire room through the window and the outdoor wind was generated by the fan and a static pressure box (cf. Figure 1). The velocity of the outdoor wind was adjusted by changing the AC frequency of the frequency converter. The velocity of the outdoor wind varied from 0 to 7.0 m/s and the corresponding full-scale outdoor wind velocity range was 0–12.12 m/s according to Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 Figure 1. Schematic view of the experimental corridor and fire room. concept (EDC), and a thermal radiation model based on a gray gas model for the The models are based on the numerical solving of Navier-Stokes equations. These equations calculate mass, momentum, species, and energy conservation [25]: > ∂ ∂xj μ ∂ui ∂xj þ ∂uj ∂xi � 2 3 δij ∂ui ∂xi ð Þ ρD <sup>k</sup> <sup>∗</sup> ∂xj <sup>T</sup> <sup>þ</sup><sup>X</sup> <sup>∂</sup> where Eq. (14) represents the mass conservation equation, Eq. (15) represents the momentum conservation equation, Eq. (16) represents the species conservation The modelling was carried out using the Deardorff turbulence model and extinction model based on a critical flame temperature. The combustion model is based on the finite rate combustion using Arrhenius parameters (A: pre-exponential factor and Ea: activation energy). The fire source was modelled as a gas burner using butane as fuel with mass flux given by the experimental data [8]. The com- The experimental setup used as reference in the current numerical study represents a reduced scale (1:3) of a facility which contains a corridor and a fire room [8]. As shown in Figure 1, the dimensions of the corridor were 5.5 m (length) � 0.7 m (width) � 0.9 m (height) and the dimensions of the fire room were 2.0 m (length) � 1.7 m (width) � 1.0 m (height). The corridor and fire room were connected by a door whose dimensions were 0.7 m long by 0.3 m wide. The window in the fire room was opposite to the door and its dimensions were 0.5 m (width) � 0.5 m (height). The ceilings and floors of the corridor and fire room were made of steel As shown in Figure 1, the fire source was in the middle of the fire room, and it was defined as a gas burner using liquefied petroleum gas as fuel. The fuel supply rates of the gas burner were controlled and monitored by a flow meter. The HRR in the experiments was determined by multiplying the mass flow rate and the combustion heat of liquefied petroleum gas. The fire size can be scaled up to 96.2 kW of The wind can blow into the fire room through the window and the outdoor wind was generated by the fan and a static pressure box (cf. Figure 1). The velocity of the outdoor wind was adjusted by changing the AC frequency of the frequency converter. The velocity of the outdoor wind varied from 0 to 7.0 m/s and the corresponding full-scale outdoor wind velocity range was 0–12.12 m/s according to ∂ ∂xj Yk <sup>∗</sup> � � � � <sup>¼</sup> <sup>0</sup> (14) þ ω\_ <sup>000</sup> � � ∂ ∂xj Yk <sup>∗</sup> (15) (17) <sup>k</sup> <sup>∗</sup> (16) � ∂ ∂xj q\_ 00 r,j � � � � hk <sup>∗</sup> ð Þ ρD <sup>k</sup> <sup>∗</sup> ∂ρ ∂t þ ∂ ∂xj ρuj þ ρgi þ ∂x<sup>2</sup> j ∂xj ∂xi dt <sup>þ</sup> <sup>q</sup>\_<sup>000</sup> <sup>þ</sup> <sup>λ</sup> <sup>∂</sup><sup>2</sup> <sup>ρ</sup>ujYk <sup>∗</sup> <sup>¼</sup> <sup>∂</sup> equation, and Eq. (17) represents the energy conservation equation. radiation absorption coefficient [15, 16]. Fire Safety and Management Awareness ∂ ∂xj ρuiuj � � ¼ � <sup>∂</sup><sup>p</sup> ∂ρYk <sup>∗</sup> ∂t þ ∂ ∂xj ð Þ¼ <sup>ρ</sup>uih dp bustion heat of butane is 45182.83 kJ/kg. plate with a thickness of 2.5 mm. 16 HRR, which corresponds to 1.5 MW full-scale. 3.3 Computational domain and boundary conditions ∂ <sup>∂</sup><sup>t</sup> <sup>ρ</sup>uj � � <sup>þ</sup> ð Þþ <sup>ρ</sup><sup>h</sup> <sup>∂</sup> ∂xj 3.2 Fire modelling ∂ ∂t the scaling law of Froude modelling [12, 13]. Varying the wind velocity, eight experiments were conducted at an HRR of 96.2 kW, equaling 1.5 MW at full-scale. The experiments were carried out with ambient temperature ranging from 6 to 16°C. K-type thermocouples with an accuracy of �1°C were used for the temperature measurements in the corridor and fire room. Hot wire wind speed meters were applied to measure the velocity of smoke. In order to model the geometry and the boundary conditions of the setup used during the experimental tests [8], the walls of the corridor and fireroom were made of steel having a density of 7850 kg/m<sup>3</sup> , a thermal conductivity of 46 W/(m�K)�<sup>1</sup> , a specific heat of 0.5 kJ/(kg�K), and an emissivity of 0.9. In simulations, the boundary condition at the window was modeled as an opening in the case without wind. With wind, a constant flow rate was set at the window using the velocity boundary used in the CFD code. In order to remain consistent with the experimental tests, a waiting time of 150 s was defined before activation of the outdoor wind velocity in the modelling. The simulations were performed in eight cases (Vw = 0, 1, 2, 3, 4, 5, 6, and 7 m/s, which correspond to Vw = 0, 1.73, 3.46, 5.20, 6.93, 8.66, 10.39, and 12.12 m/s full-scale). Similarly, the simulation results were converted into full-scale data according to the Froude number. The smoke temperature, smoke velocity, concentration of O2 and CO, and visibility in the corridor were analyzed by setting different devices in the plane (Y = 0.5 m), near the exit of the corridor (X = 5.4 m), at different heights (Z = 85, 70, 55, 40, and 25 cm). Moreover, other observations were carried out about the distribution of temperature, velocity, concentration of O2 and CO2, and visibility thanks to slice fields in the plane Y = 0.5 m. #### 3.4 Mesh size resolution For numerical studies, it is important to choose the correct mesh size in order to obtain accurate simulation results. FDS provides a range of mesh sizes for mesh resolution. From a Poisson solver based on the fast Fourier transform (FFT), it is possible to obtain good numerical resolution by solving the governing equations. The mesh size was chosen in accordance with the recommendations made in the numerical studies [15, 16]. An optimal mesh size should meet two requirements: good results in terms of accuracy and a short calculation time. The optimal mesh size of the domain is given by the nondimensional expression <sup>D</sup><sup>∗</sup> =<sup>∂</sup>x, where ∂x is the nominal mesh size and D\* is the characteristic fire diameter [24]. The characteristic fire diameter D\* is determined using Eq. (18): $$\mathbf{D}^\* = \left(\frac{\dot{\mathbf{Q}}}{\rho\_{\text{osc}\_{\text{P}}\text{T}\_{\text{ov}}\sqrt{\mathbf{g}}}}\right)^{\sharp \boldsymbol{\xi}} \tag{18}$$ where D\* denotes the characteristic fire diameter, Q the Heat Release Rate, and \_ cp the specific heat. Based on several experiments, the U.S. Nuclear Regulatory Commission recommends that the numerical range of <sup>D</sup><sup>∗</sup> =<sup>∂</sup><sup>x</sup> be between 4 and 16 for simulations to produce favorable results at a moderate computational cost, since the larger the value of <sup>D</sup><sup>∗</sup> =<sup>∂</sup><sup>x</sup> used in the simulation, the more accurate the simulation result. Hence, the range of mesh sizes can be obtained by the following equation [15, 16]: $$\frac{\mathbf{D}^\}{16} \le \delta\_\mathbf{x} \le \frac{\mathbf{D}^\}{4} \tag{19}$$ reinforced by the suggestions of the various cases of validation of the FDS code Numerical grid Number of cells Relative gap (%) CPU time (h) Mesh size 20 cm 1685 31.06 33.17 1.2 Mesh size 10 cm 11865 19.76 16.91 4.4 Mesh size 5 cm 94920 6.06 6.23 9.6 Mesh size 2.5 cm 759360 3.80 3.85 92.2 Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor Temperature (°C) Smoke velocity (m/s) With the 5-cm mesh, the total number of cells is 94920 and the simulation time is 1000 s with a time step of 0.010 s. The calculations were carried out using 20 processors in the ARTEMIS cluster of the "Région Centre Val de Loire—France" Figure 2(a) and (b) shows that the numerical results obtained with the 5-cm mesh are in agreement with experimental data as regards the evolution of smoke temperature and smoke velocity [8]. This indicates that with a 5-cm mesh, the boundary conditions can be satisfactorily modeled by FDS and that the interaction Smoke velocity with: (a) Vw = 1.73 m/s; (b) Vw = 3.46 m/s; (c) Vw = 6.93 m/s; and (d) Vw = 12.12 m/s at a proposed in the user guide [24]. Results of different numerical grid mesh sizes. DOI: http://dx.doi.org/10.5772/intechopen.92978 Table 1. Figure 3. 19 height of 70 cm of FDS and experimental results [8]. and each computation took about 9.6 h. between wind and smoke flow can be reproduced. After calculation, the range of mesh sizes was found to be: (0.0625 and 0.25 m). Therefore, four different mesh sizes were used: 20, 10, 5, and 2.5 cm. Figure 2(a) and (b) presents the comparisons between experiment and FDS predictions for these four different meshes. The comparisons were carried out on the evolution of the smoke temperature and smoke velocity, both measured 70 cm above the ground and in the centerline of the corridor near the exit. It can be seen that the numerical results obtained with mesh sizes of 5 and 2.5 cm converge with the experimental results, while the results of the 20 and 10 cm meshes diverge. Moreover, the 2.5-cm mesh gives more accurate numerical results than the 5-cm mesh. As shown in Table 1, the relative gap (RG) of the calculation with the 2.5-cm mesh (3.85%) is slightly smaller than the calculation with a 5-cm mesh (6.83%). The relative gap (RG) is obtained by [26]: $$\text{RG} = \mathbf{100} \times \frac{\sqrt{\sum\_{i=1}^{n} \left(\mathbf{y}\_{\text{pre},i} - \mathbf{y}\_{\text{exp},i}\right)^2}}{\sqrt{\sum\_{i=1}^{n} \left(\mathbf{y}\_{\text{exp},i}\right)^2}} \tag{20}$$ where ypre is a predicted value, yexp is an experimental value, and n is the number of experimental points. However, the calculation time with the 2.5-cm mesh is 10 times longer than with the 5-cm mesh. In addition, the relative gap of the 5-cm mesh is close to that of the 2.5-cm mesh. As it represents the best trade-off between precision and calculation time, the 5-cm mesh was used for the following calculations, this choice is Figure 2. The influence of grid cells on: (a) temperature at a height of 70 cm; and (b) smoke velocity without wind at a height of 70 cm. Table 1. where D\* denotes the characteristic fire diameter, Q the Heat Release Rate, and \_ Based on several experiments, the U.S. Nuclear Regulatory Commission recom- <sup>∂</sup><sup>x</sup> used in the simulation, the more accurate the simulation result. Hence, the range of mesh sizes can be obtained by the following equation [15, 16]: After calculation, the range of mesh sizes was found to be: (0.0625 and 0.25 m). It can be seen that the numerical results obtained with mesh sizes of 5 and 2.5 cm ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi <sup>i</sup>¼<sup>1</sup> ypre,i � <sup>y</sup>exp ,i � �<sup>2</sup> <sup>r</sup> ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi � �<sup>2</sup> <sup>r</sup> (20) <sup>i</sup>¼<sup>1</sup> <sup>y</sup>exp ,i D<sup>∗</sup> <sup>∂</sup><sup>x</sup> be between 4 and 16 for simulations to <sup>4</sup> (19) = D<sup>∗</sup> <sup>16</sup> <sup>≤</sup>δ<sup>x</sup> <sup>≤</sup> Therefore, four different mesh sizes were used: 20, 10, 5, and 2.5 cm. cm above the ground and in the centerline of the corridor near the exit. mesh (6.83%). The relative gap (RG) is obtained by [26]: RG ¼ 100 � number of experimental points. Figure 2. 18 height of 70 cm. converge with the experimental results, while the results of the 20 and 10 cm meshes diverge. Moreover, the 2.5-cm mesh gives more accurate numerical results than the 5-cm mesh. As shown in Table 1, the relative gap (RG) of the calculation with the 2.5-cm mesh (3.85%) is slightly smaller than the calculation with a 5-cm P<sup>n</sup> where ypre is a predicted value, yexp is an experimental value, and n is the The influence of grid cells on: (a) temperature at a height of 70 cm; and (b) smoke velocity without wind at a time, the 5-cm mesh was used for the following calculations, this choice is P<sup>n</sup> However, the calculation time with the 2.5-cm mesh is 10 times longer than with the 5-cm mesh. In addition, the relative gap of the 5-cm mesh is close to that of the 2.5-cm mesh. As it represents the best trade-off between precision and calculation Figure 2(a) and (b) presents the comparisons between experiment and FDS predictions for these four different meshes. The comparisons were carried out on the evolution of the smoke temperature and smoke velocity, both measured 70 produce favorable results at a moderate computational cost, since the larger the cp the specific heat. = value of <sup>D</sup><sup>∗</sup> mends that the numerical range of <sup>D</sup><sup>∗</sup> Fire Safety and Management Awareness Results of different numerical grid mesh sizes. reinforced by the suggestions of the various cases of validation of the FDS code proposed in the user guide [24]. With the 5-cm mesh, the total number of cells is 94920 and the simulation time is 1000 s with a time step of 0.010 s. The calculations were carried out using 20 processors in the ARTEMIS cluster of the "Région Centre Val de Loire—France" and each computation took about 9.6 h. Figure 2(a) and (b) shows that the numerical results obtained with the 5-cm mesh are in agreement with experimental data as regards the evolution of smoke temperature and smoke velocity [8]. This indicates that with a 5-cm mesh, the boundary conditions can be satisfactorily modeled by FDS and that the interaction between wind and smoke flow can be reproduced. #### Figure 3. Smoke velocity with: (a) Vw = 1.73 m/s; (b) Vw = 3.46 m/s; (c) Vw = 6.93 m/s; and (d) Vw = 12.12 m/s at a height of 70 cm of FDS and experimental results [8]. Figure 3 plots the smoke velocity decays with different wind velocities: (a) Vw = 1.73 m/s; (b) Vw = 5.20 m/s; (c) Vw = 6.93 m/s; and (d) Vw = 10.93 m/s at 70 cm height. It can be seen that the predictions of the evolution of smoke velocities are similar to those of the experimental data [8]. Since the velocities were measured at a height of 70 cm in the experiments, these values are in fact averages. Therefore, it is possible that for some values of the smoke velocity, the experimental data are underestimated or overestimated. In these conditions, predictions are overestimated at the start or at the end of the curves Figure 3(a) and (b). These small differences can be associated to the vortex waves that are not very well reproduced by the turbulence model. To try to improve it, a sensibility analysis can be performed on the different turbulence models [24]. However, good agreement between prediction and experiment is observed in the other pictures (Figure 3(c) and (d)). It can be concluded from these different comparisons that the choice of a 5-cm mesh is suitable and that it can deal with reactive flows with a good accuracy. Leakage was neglected during the modelling, as the amount of leakage in the experiment is unknown. It is possible, therefore, that some simulation results may be under- or overestimated. Overall, however, the predictions of the simulations are acceptable. #### 4. Results and discussions In this part of the chapter, the numerical results with different wind velocities (Vw = 1.73, 3.46, and 5.20 m/s) are discussed in terms of the effects of outdoor wind on smoke stratification and smoke extraction. A global sensitivity analysis was carried out in order to determine the effects of the input parameters on the output data. The target input parameters are mass flux (MF) of fuel, the material properties (conductivity λ, emissivity ε, density ρ, and specific heat cp), and the Arrhenius parameters (A, Ea). The target output data are the smoke temperature near the ceiling. #### 4.1 Outdoor wind effect on the smoke exhaust Figure 4 presents the smoke velocity field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s; in the cross-section y = 0.5 m at 300 s. The cross-section y = 0.5 m is the plane in the middle of the corridor. In Figure 4(a), taking this plane at the height of 70 cm, the maximum value of the smoke velocity is near the door and decreases with the distance from the door as shown in Figures 3 and 4. In addition, considering smoke stratification with a hot zone near the ceiling and a cold zone near the floor, it is observed that the buoyancy effects give the reverse observation. Near the floor, the smoke velocity increases with the distance, and using the vortex recirculation solved by the Deardorff turbulence model, the numerical solver can reproduce the vortex flow induced by the smoke flow. From Figure 4, the maximum of smoke velocity in the corridor increases when the wind velocity increases. As mentioned previously, in these conditions, smoke exhaust can be disturbed. Outdoor wind can, however, contribute to the evacuation of smoke and fire extinction in that more smoke is extracted through the corridor when the wind velocity increases. It should nevertheless be mentioned that while ventilation and extraction systems play an important role in fire engineering [15], the efficiency of the smoke extraction system will be reduced and even be invalidated when the outdoor wind velocity is very high and the extraction system is installed in the windward surface of the compartment [21]. In this case, the extraction system cannot perform well, and smoke can spread along the entire compartment through the connected rooms. This situation is not acceptable Simulation of the smoke velocity field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 and (d) Vw = 5.20 m/s in the cross-section y = 0.5 m at 300 s. Figure 5 shows that when the outdoor wind velocity increases, the oxygen concentration increases and carbon dioxide concentration decreases. Figure 5 presents the influence of wind velocity on O2 concentration and CO2 concentration at a for fire safety. Figure 4. 21 Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 Figure 4. Figure 3 plots the smoke velocity decays with different wind velocities: (a) Vw = 1.73 m/s; (b) Vw = 5.20 m/s; (c) Vw = 6.93 m/s; and (d) Vw = 10.93 m/s at 70 cm height. It can be seen that the predictions of the evolution of smoke velocities are similar to those of the experimental data [8]. Since the velocities were measured at a Therefore, it is possible that for some values of the smoke velocity, the experimental data are underestimated or overestimated. In these conditions, predictions are overestimated at the start or at the end of the curves Figure 3(a) and (b). These small differences can be associated to the vortex waves that are not very well reproduced by the turbulence model. To try to improve it, a sensibility analysis can be performed on the different turbulence models [24]. However, good agreement between prediction It can be concluded from these different comparisons that the choice of a 5-cm In this part of the chapter, the numerical results with different wind velocities (Vw = 1.73, 3.46, and 5.20 m/s) are discussed in terms of the effects of outdoor wind on smoke stratification and smoke extraction. A global sensitivity analysis was carried out in order to determine the effects of the input parameters on the output data. The target input parameters are mass flux (MF) of fuel, the material properties (conductivity λ, emissivity ε, density ρ, and specific heat cp), and the Arrhenius parameters (A, Ea). The target output data are the smoke temperature near the Figure 4 presents the smoke velocity field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s; in the cross-section y = 0.5 m at 300 s. The cross-section y = 0.5 m is the plane in the middle of the corridor. In Figure 4(a), taking this plane at the height of 70 cm, the maximum value of the smoke velocity is near the door and decreases with the distance from the door as shown in Figures 3 and 4. In addition, considering smoke stratification with a hot zone near the ceiling and a cold zone near the floor, it is observed that the buoyancy effects give the reverse observation. Near the floor, the smoke velocity increases with the distance, and using the vortex recirculation solved by the Deardorff turbulence model, the numerical solver can reproduce the vortex flow induced by From Figure 4, the maximum of smoke velocity in the corridor increases when the wind velocity increases. As mentioned previously, in these conditions, smoke exhaust can be disturbed. Outdoor wind can, however, contribute to the evacuation of smoke and fire extinction in that more smoke is extracted through the corridor when the wind velocity increases. It should nevertheless be mentioned that while ventilation and extraction systems play an important role in fire engineering [15], the efficiency of the smoke extraction system will be reduced and even be invalidated when the outdoor wind velocity is very high and the extraction system is installed in the windward surface of the compartment [21]. In this mesh is suitable and that it can deal with reactive flows with a good accuracy. Leakage was neglected during the modelling, as the amount of leakage in the experiment is unknown. It is possible, therefore, that some simulation results may be under- or overestimated. Overall, however, the predictions of the simulations are height of 70 cm in the experiments, these values are in fact averages. and experiment is observed in the other pictures (Figure 3(c) and (d)). acceptable. ceiling. the smoke flow. 20 4. Results and discussions Fire Safety and Management Awareness 4.1 Outdoor wind effect on the smoke exhaust Simulation of the smoke velocity field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s in the cross-section y = 0.5 m at 300 s. case, the extraction system cannot perform well, and smoke can spread along the entire compartment through the connected rooms. This situation is not acceptable for fire safety. Figure 5 shows that when the outdoor wind velocity increases, the oxygen concentration increases and carbon dioxide concentration decreases. Figure 5 presents the influence of wind velocity on O2 concentration and CO2 concentration at a Figure 5. Influence of wind velocity on: (a) O2 concentration; and (b) CO2 concentration at 85 cm height. height of 85 cm (on the ceiling of the corridor), showing that the more wind velocity increases, the more oxygen concentration increases. After 300 s, the oxygen concentration remains stable when the wind velocity varies from 0 to 6.93 m/s. The oxygen concentration at 300 s was therefore used to compare the different wind velocity cases. is so small that it would have little effect on people's health, the homogeneous distribution of CO concentration may cause serious problems when the heat release Influence of wind velocity on: (a) CO concentration; and (b) visibility at 55 cm height at the exit of the Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 the corridor gradually becomes homogeneous as the outdoor wind velocity increases and becomes better when the wind velocity reaches 5.20 m/s, indicating In other words, smoke can exit the corridor faster when the wind velocity increases. It can be said that to some extent, the outdoor wind is helpful for smoke exhaust and an advantage for the evacuation of people in fires as it can decrease the concentration of toxic gas and improve visibility in the environment. However, in these conditions, the outdoor wind becomes a disturbance for the extraction sys- 4.2 Outdoor wind effect on the smoke stratification and sensitivity analysis In Li et al. [8], it was shown that the more wind velocity increased, the more severely the smoke stratification was disturbed. This observation was obtained by comparing the smoke temperature near the floor (height = 25 cm) and the smoke temperature near the ceiling (height = 85 cm). The tests were performed for three velocities. The results showed that above a wind velocity of 3.46 m/s, the smoke temperatures near the floor and the ceiling were similar. This similarity was taken to imply that the smoke occupied the entire corridor volume, due to the absence of smoke stratification, and the numerical data used in the current study confirmed Thanks to Figure 7, it is possible to make a comparison between the smoke temperature near the ceiling and near the floor. It is constated that the more wind speed increases, the more the smoke stratification is disturbed. Smoke stratification is represented by the stability between the hot zone and the cold zone. The hot zone is formed by hot smoke and the cold zone is formed by cold air. Smoke stratification in an enclosure is due to the temperature difference between these two zones. In addition, as shown in the literature [27–29], smoke stratification depends on the Froude number. Smoke stratification is very stable up to a critical Froude number that the more smoke is exhausted, the more visibility is improved. tem, representing an unacceptable situation for fire safety. this observation. 23 Figure 6. corridor. Moreover, concerning the visibility, it is shown that the visibility of the lower area in the corridor is very high and the visibility of the upper area in the corridor is very low due to smoke stratification when there is no wind. For this, the visibility in rate in the building is larger, producing more CO. When the wind velocity is 1.73 m/s, the O2 molar concentration increases only slightly compared to a situation without wind. When the wind velocity increases to 3.46 m/s, the O2 molar concentration increases strongly compared to the case without wind. For a wind velocity of 6.93 m/s, the O2 molar concentration increases to 20.2%, 1.8% higher than without wind. The rise in O2 concentration in the corridor also indicates that more smoke is extracted. The more the wind velocity increases, the more the CO2 concentration decreases (Figure 5(b)). At a wind velocity of 6.93 m/s, the CO2 molar concentration decreases to 0.3%, 1% lower than without wind. The decline of the CO2 concentration in the corridor also contributes to people escaping from fires. Using oxygen concentration field like the smoke velocity field in the Figure 4, the mean oxygen concentration in the corridor increases when the wind velocity increases, showing that a higher wind velocity can facilitate smoke exhaust. It is also possible to highlight the influence of wind velocity on CO concentration and visibility. The evolutions of these latest are presented in Figure 6 at a height of 50 cm. The height of 50 cm represents the average height of a person measuring 165 cm in a full-scale building. From Figure 6(a), CO concentration decreases with wind velocity. The outdoor wind can thus be an advantage for diluting the CO concentration. Figure 6(b) shows that the more the outdoor wind velocity increases, the more the visibility increases. Thus, the more wind blows in, the more smoke is diluted. However, the visibility becomes homogenous in the enclosure due to disturbance in the smoke stratification. In a fire with a heat release rate larger than the one used in this study, the poor visibility can be unfavorable for the evacuation of people in the building. Using the CO concentration field similarly to the smoke filed, the average concentration of CO decreases with the increase in wind velocity. There are two zones: a thin zone near the floor and a thick zone near the ceiling in the case of no wind. The distribution of CO concentration in the corridor gradually becomes homogeneous as the wind velocity increases. Although in this study the CO concentration Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 Figure 6. Influence of wind velocity on: (a) CO concentration; and (b) visibility at 55 cm height at the exit of the corridor. is so small that it would have little effect on people's health, the homogeneous distribution of CO concentration may cause serious problems when the heat release rate in the building is larger, producing more CO. Moreover, concerning the visibility, it is shown that the visibility of the lower area in the corridor is very high and the visibility of the upper area in the corridor is very low due to smoke stratification when there is no wind. For this, the visibility in the corridor gradually becomes homogeneous as the outdoor wind velocity increases and becomes better when the wind velocity reaches 5.20 m/s, indicating that the more smoke is exhausted, the more visibility is improved. In other words, smoke can exit the corridor faster when the wind velocity increases. It can be said that to some extent, the outdoor wind is helpful for smoke exhaust and an advantage for the evacuation of people in fires as it can decrease the concentration of toxic gas and improve visibility in the environment. However, in these conditions, the outdoor wind becomes a disturbance for the extraction system, representing an unacceptable situation for fire safety. #### 4.2 Outdoor wind effect on the smoke stratification and sensitivity analysis In Li et al. [8], it was shown that the more wind velocity increased, the more severely the smoke stratification was disturbed. This observation was obtained by comparing the smoke temperature near the floor (height = 25 cm) and the smoke temperature near the ceiling (height = 85 cm). The tests were performed for three velocities. The results showed that above a wind velocity of 3.46 m/s, the smoke temperatures near the floor and the ceiling were similar. This similarity was taken to imply that the smoke occupied the entire corridor volume, due to the absence of smoke stratification, and the numerical data used in the current study confirmed this observation. Thanks to Figure 7, it is possible to make a comparison between the smoke temperature near the ceiling and near the floor. It is constated that the more wind speed increases, the more the smoke stratification is disturbed. Smoke stratification is represented by the stability between the hot zone and the cold zone. The hot zone is formed by hot smoke and the cold zone is formed by cold air. Smoke stratification in an enclosure is due to the temperature difference between these two zones. In addition, as shown in the literature [27–29], smoke stratification depends on the Froude number. Smoke stratification is very stable up to a critical Froude number height of 85 cm (on the ceiling of the corridor), showing that the more wind velocity increases, the more oxygen concentration increases. After 300 s, the oxygen concentration remains stable when the wind velocity varies from 0 to 6.93 m/s. The oxygen concentration at 300 s was therefore used to compare the different Influence of wind velocity on: (a) O2 concentration; and (b) CO2 concentration at 85 cm height. corridor also indicates that more smoke is extracted. When the wind velocity is 1.73 m/s, the O2 molar concentration increases only slightly compared to a situation without wind. When the wind velocity increases to 3.46 m/s, the O2 molar concentration increases strongly compared to the case without wind. For a wind velocity of 6.93 m/s, the O2 molar concentration increases to 20.2%, 1.8% higher than without wind. The rise in O2 concentration in the The more the wind velocity increases, the more the CO2 concentration decreases Using oxygen concentration field like the smoke velocity field in the Figure 4, the mean oxygen concentration in the corridor increases when the wind velocity increases, showing that a higher wind velocity can facilitate smoke exhaust. It is also possible to highlight the influence of wind velocity on CO concentration and visibility. The evolutions of these latest are presented in Figure 6 at a height of 50 cm. The height of 50 cm represents the average height of a person measuring 165 cm in a full-scale building. From Figure 6(a), CO concentration decreases with The outdoor wind can thus be an advantage for diluting the CO concentration. Figure 6(b) shows that the more the outdoor wind velocity increases, the more the visibility increases. Thus, the more wind blows in, the more smoke is diluted. However, the visibility becomes homogenous in the enclosure due to disturbance in the smoke stratification. In a fire with a heat release rate larger than the one used in this study, the poor visibility can be unfavorable for the evacuation of people in the Using the CO concentration field similarly to the smoke filed, the average concentration of CO decreases with the increase in wind velocity. There are two zones: a thin zone near the floor and a thick zone near the ceiling in the case of The distribution of CO concentration in the corridor gradually becomes homogeneous as the wind velocity increases. Although in this study the CO concentration (Figure 5(b)). At a wind velocity of 6.93 m/s, the CO2 molar concentration decreases to 0.3%, 1% lower than without wind. The decline of the CO2 concentra- tion in the corridor also contributes to people escaping from fires. wind velocity cases. Fire Safety and Management Awareness Figure 5. wind velocity. building. no wind. 22 3.46 m/s, smoke stratification almost disappears, as the smoke temperature is similar at the different heights and only the hot zone subsists. In this condition, smoke occupies the entire corridor. The phenomenon of temperature stratification in the Smoke temperature field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; (d) Vw = 5.20 m/s on corridor disappears completely when the wind velocity reaches 5.20 m/s (Figure 8(d)), as also shown with the curves in Figure 7(d). These results also show that thanks to simulations performed by FDS, it is possible to demonstrate the Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor fields of smoke movement in an enclosure with outdoor wind. DOI: http://dx.doi.org/10.5772/intechopen.92978 Figure 8. 25 the cross-section y = 0.5 m at 300 s. Figure 7. Influence of wind velocity (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s on smoke temperature in different heights of the corridor. and becomes disturbed when the Froude number is larger than this critical value. It is known that the Froude number can be associated to velocity. So, smoke stratification is related to the smoke velocity in an enclosure. In Figure 7(a), when the wind velocity is 0 m/s, the smoke temperature near the ceiling and floor are about 5 and 60°C, indicating that smoke stratification is very stable. At a wind velocity of 1.73 m/s, the smoke temperature near the ceiling and floor are about 25 and 100°C, respectively. In this condition, there are still two zones. In Figure 7(b), there is a slight perturbation of the temperature near the ceiling, indicating a slight disturbance in the smoke stratification. However, from a wind velocity of 3.46 m/s, the smoke temperature near the ceiling and floor are similar with an average of 80°C. This means that above this velocity, there is no smoke stratification in the corridor and that smoke occupies the entire corridor. Under these conditions, there is a risk of toxicity for people. These observations confirm results reported in the literature [8] and highlight the ability of the CFD code to reproduce the effects of wind on the movement of smoke in an enclosure. Moreover, in these conditions, outdoor wind becomes a disturbance for smoke extraction, creating an unacceptable situation for fire safety. The smoke temperature field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s in the cross-section y = 0.5 m at 300 s is shown in Figure 8. It can be clearly seen that when there is no outdoor wind, there is temperature stratification in the corridor. In addition, the temperature near the ceiling is much higher than the temperature near the floor. For a wind velocity of 1.73 m/s, smoke stratification is disturbed but still exists due to the presence of two zones, with a much smaller cold zone than hot zone. When the wind velocity is Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 3.46 m/s, smoke stratification almost disappears, as the smoke temperature is similar at the different heights and only the hot zone subsists. In this condition, smoke occupies the entire corridor. The phenomenon of temperature stratification in the corridor disappears completely when the wind velocity reaches 5.20 m/s (Figure 8(d)), as also shown with the curves in Figure 7(d). These results also show that thanks to simulations performed by FDS, it is possible to demonstrate the fields of smoke movement in an enclosure with outdoor wind. #### Figure 8. Smoke temperature field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; (d) Vw = 5.20 m/s on the cross-section y = 0.5 m at 300 s. and becomes disturbed when the Froude number is larger than this critical value. It is known that the Froude number can be associated to velocity. So, smoke stratification is related to the smoke velocity in an enclosure. In Figure 7(a), when the wind velocity is 0 m/s, the smoke temperature near the ceiling and floor are about 5 and 60°C, indicating that smoke stratification is very stable. At a wind velocity of 1.73 m/s, the smoke temperature near the ceiling and floor are about 25 and 100°C, respectively. In this condition, there are still two zones. In Figure 7(b), there is a slight perturbation of the temperature near the ceiling, indicating a slight disturbance in the smoke stratification. However, from a wind velocity of 3.46 m/s, the smoke temperature near the ceiling and floor are similar with an average of 80°C. This means that above this velocity, there is no smoke stratification in the corridor and that smoke occupies the entire corridor. Under these conditions, there is a risk of toxicity for people. These observations confirm results reported in the literature [8] and highlight the ability of the CFD code to reproduce the effects of wind on the movement of smoke in an enclosure. Moreover, in these conditions, outdoor wind becomes a disturbance for smoke extraction, creating an unacceptable situation for Influence of wind velocity (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s on The smoke temperature field with (a) Vw = 0 m/s; (b) Vw = 1.73 m/s; (c) Vw = 3.46 m/s; and (d) Vw = 5.20 m/s in the cross-section y = 0.5 m at 300 s is shown in Figure 8. It can be clearly seen that when there is no outdoor wind, there is temperature stratification in the corridor. In addition, the temperature near the ceiling is much higher than the temperature near the floor. For a wind velocity of 1.73 m/s, smoke stratification is disturbed but still exists due to the presence of two zones, with a much smaller cold zone than hot zone. When the wind velocity is fire safety. 24 Figure 7. smoke temperature in different heights of the corridor. Fire Safety and Management Awareness compartment fire. The focus was on the effects of outdoor wind on the dynamics of smoke spreading based on experimental data. Simulations were carried out by varying wind velocity from 0 to 12.12 m/s. Good agreement between experimental data and prediction was found, enabling investigation of smoke stratification, smoke exhaust, and a global sensitivity analysis. The major findings include the Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor 1.By analyzing the temperature distribution in the corridor, it was found that smoke stratification can be strongly affected by the outdoor wind. For wind velocities higher than the critical value of 3.46 m/s, smoke stratification is 2.When the wind velocity is higher than the critical value (here 3.46 m/s), O2 concentration and visibility increase, while CO2 and CO concentration tends to decrease. It is shown that the magnitude of the outdoor wind can facilitate 3.The results of a global sensitivity analysis indicate that it is essential to define the most influential input parameters correctly, namely the mass flux of the fuel and the activation energy. If not, large deviations in the outputs of the numerical results such as smoke temperature may occur due to variations, 4.Based on the amplitude of the metamodel coefficients, a reduced metamodel has been proposed. A prediction with a confidence interval can be easily implemented, leading to close agreement with the numerical results. Through this work, it is demonstrated that CFD FDS can provide information about the movement of smoke in a corridor. Besides, it can be coupled with a polynomial chaos-based sensitivity analysis, which enables the input parameters to In addition, considering the importance of the effects of outside wind on reac- be classified on quantitative grounds with a limited computational cost. tive flows induced by a fire in a building, it is important to study other study configurations. In this context, it would be important to also study the role of the outside wind on the ignition of smoke rich in unburnt gas in the case of an following: completely disturbed. DOI: http://dx.doi.org/10.5772/intechopen.92978 under-ventilated fire. 27 smoke exhaust in a compartment fire. even slight ones, in the input parameters. #### Figure 9. Local sensitivity indices. In addition, from a global sensitivity analysis, an investigation was carried out in order to highlight the relative importance of seven parameters: mass flux MF, activation energy Ea, conductivity λ, emissivity ϵ, pre-exponential factor A, density ρ, and specific heat cp. The aim was to determine whether, among these seven parameters, even a slight modification of the input parameter may cause a large variation in the response. The quantity of interest is the temperature near the ceiling. A tolerance interval of �10% was applied to each of the inputs so that for each of the inputs, a dimensionless random parameter is introduced. Its value depends on the realization θ and belongs to the interval [�1,1]. In this context, the values of the random parameters MF and Ea depend on their mean value MF and Ea, on the tolerance interval �10%, and on the random dimensionless parameter ξMF and ξEa (whose values depend on the observation θ). For that, an analytical model is proposed to predict the time evolution of the quantity of interest for arbitrary values of mass flux (MF) and activation energy (Ea), such that: $$\mathbf{MF}(\boldsymbol{\theta}) = \overline{\mathbf{MF}}(\mathbf{1} + \mathbf{0}.\mathbf{1} \,\mathsf{\xi}\_{\mathrm{MF}}(\boldsymbol{\theta}))$$ $$\mathbf{E}\_{\mathfrak{a}}(\boldsymbol{\theta}) = \overline{\mathbf{E}\_{\mathfrak{a}}}(\mathbf{1} + \mathbf{0}.\mathbf{1} \,\mathsf{\xi}\_{\mathrm{E}\_{\mathfrak{a}}}(\boldsymbol{\theta})) \tag{21}$$ Indeed, using the smoke temperature as out data based on the methodology of the sensitivity analysis proposed by Chaos [20], Figure 9 shows that the mass flux of the fuel and the activation energy are the two parameters which are an important influence on the smoke temperature. Moreover, Figure 9 presents the first-order sensitivity and the total sensitivity indices. Considering this influence, it is very important to define the values of the mass flux of the fuel and the activation energy with a good accuracy in order to over or underestimate the out data such as the temperature, heat flux, pressure, and the amount of species. #### 5. Conclusion In this chapter, a CFD code, namely fire dynamics simulator (FDS), was employed to model the smoke spreading along a corridor induced by a #### Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978 compartment fire. The focus was on the effects of outdoor wind on the dynamics of smoke spreading based on experimental data. Simulations were carried out by varying wind velocity from 0 to 12.12 m/s. Good agreement between experimental data and prediction was found, enabling investigation of smoke stratification, smoke exhaust, and a global sensitivity analysis. The major findings include the following: Through this work, it is demonstrated that CFD FDS can provide information about the movement of smoke in a corridor. Besides, it can be coupled with a polynomial chaos-based sensitivity analysis, which enables the input parameters to be classified on quantitative grounds with a limited computational cost. In addition, considering the importance of the effects of outside wind on reactive flows induced by a fire in a building, it is important to study other study configurations. In this context, it would be important to also study the role of the outside wind on the ignition of smoke rich in unburnt gas in the case of an under-ventilated fire. In addition, from a global sensitivity analysis, an investigation was carried out in In this context, the values of the random parameters MF and Ea depend on their mean value MF and Ea, on the tolerance interval �10%, and on the random dimensionless parameter ξMF and ξEa (whose values depend on the observation θ). For that, an analytical model is proposed to predict the time evolution of the quantity of interest for arbitrary values of mass flux (MF) and activation energy (Ea), such that: MFð Þ¼ θ MF 1ð Þ þ 0:1 ξMFð Þθ Indeed, using the smoke temperature as out data based on the methodology of the sensitivity analysis proposed by Chaos [20], Figure 9 shows that the mass flux of the fuel and the activation energy are the two parameters which are an important Moreover, Figure 9 presents the first-order sensitivity and the total sensitivity indices. Considering this influence, it is very important to define the values of the mass flux of the fuel and the activation energy with a good accuracy in order to over or underestimate the out data such as the temperature, heat flux, pressure, and the In this chapter, a CFD code, namely fire dynamics simulator (FDS), was employed to model the smoke spreading along a corridor induced by a ð Þ<sup>θ</sup> (21) Eað Þ¼ θ Ea 1 þ 0:1 ξEa order to highlight the relative importance of seven parameters: mass flux MF, activation energy Ea, conductivity λ, emissivity ϵ, pre-exponential factor A, density ρ, and specific heat cp. The aim was to determine whether, among these seven parameters, even a slight modification of the input parameter may cause a large variation in the response. The quantity of interest is the temperature near the ceiling. A tolerance interval of �10% was applied to each of the inputs so that for each of the inputs, a dimensionless random parameter is introduced. Its value depends on the realization θ and belongs to the interval [�1,1]. influence on the smoke temperature. amount of species. Figure 9. Local sensitivity indices. Fire Safety and Management Awareness 5. Conclusion 26 #### Author details Brady Manescau<sup>1</sup> \, Khaled Chetehouna<sup>1</sup> , Quentin Serra<sup>2</sup> , Aijuan Wang<sup>1</sup> and Eric Florentin<sup>2</sup> References* firesaf.2007.10.003 [3] Hull TR, Brein D, Stec AA. in buildings. Journal of Building Engineering. 2016;8:313-318. DOI: 10.1016/j.jobe.2016.02.014 414-421. DOI: 10.1016/j. firesaf.2017.04.037 233-243. DOI: 10.1016/j. applthermaleng.2017.01.099 857-865. DOI: 10.1016/j. applthermaleng.2016.07.141 29 [1] Yu-Ting E, Zhou L. The Research on the current safety status of high-rise building at home and abroad. Procedia Engineering. 2016;135:574-577. 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Smoke spread velocity along a 1 INSA Centre Val de Loire, University of Orléans, PRISME, Bourges, France 2 INSA Centre Val de Loire, University of Orléans, University of Tours, LaMé, Bourges, France \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Numerical Study on the Outdoor Wind Effects on Movement Smoke along a Corridor DOI: http://dx.doi.org/10.5772/intechopen.92978* #### References [1] Yu-Ting E, Zhou L. The Research on the current safety status of high-rise building at home and abroad. Procedia Engineering. 2016;135:574-577. DOI: 10.1016/j.proeng.2016.01.108 [2] Paul KT, Hull TR, Lebek K, Stec AA. Fire smoke toxicity: The effect of nitrogen oxides. 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Reliability Engineering and System Author details Fire Safety and Management Awareness Brady Manescau<sup>1</sup> Eric Florentin<sup>2</sup> Bourges, France 28 \, Khaled Chetehouna<sup>1</sup> \Address all correspondence to: [email protected] provided the original work is properly cited. , Quentin Serra<sup>2</sup> 1 INSA Centre Val de Loire, University of Orléans, PRISME, Bourges, France 2 INSA Centre Val de Loire, University of Orléans, University of Tours, LaMé, © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, , Aijuan Wang<sup>1</sup> and Safety. 2018;170:20-30. DOI: 10.1016/j. ress.2017.10.007 [15] Sellami I, Manescau B, Chetehouna K, de Izarra C, Nait-Said R, Zidani F. BLEVE fireball modeling using fire dynamics simulator (FDS) in an Algerian gas industry. Journal of Loss Prevention in the Process Industries. 2018;54. DOI: 10.1016/j.jlp.2018.02.010 [16] Magnognou B, Garo JP, Coudour B, Wang HY. Risk analysis of unburnt gas ignition in an exhaust system connected to a confined and mechanically ventilated enclosure fire. Fire Safety Journal. 2017;91:291-302. DOI: 10.1016/ j.firesaf.2017.03.036 [17] Saltelli A, Ratto M, Tarantola S, Campolongo F. Sensitivity Analysis Practice: A Guide to Scientific Models. 2006. DOI: 10.1016/j.ress.2005.11.014 [18] Sobol IM. Sensitivity analysis for nonlinear mathematical models. Mathematical Modeling and Computation. 1993;1:407-414. DOI: 10.18287/0134-2452-2015-39-4-459-461 [19] Crestaux T, Le Maître O, Martinez JM. Polynomial chaos expansion for sensitivity analysis. Reliability Engineering and System Safety. 2009;94:1161-1172. DOI: 10.1016/j.ress.2008.10.008 [20] Chaos M. Application of sensitivity analyses to condensed-phase pyrolysis modeling. Fire Safety Journal. 2013;61: 254-264. DOI: 10.1016/j.firesaf.2013. 09.016 [21] Yi L, Gao Y, Niu JL, Yang SJ. Study on effect of wind on natural smoke exhaust of enclosure fire with a twolayer zone model. Journal of Wind Engineering and Industrial Aerodynamics. 2013;119:28-38. DOI: 10.1016/j.jweia.2013.05.005 [22] Xiu D, Lucor D, Su C-H, Karniadakis GE. Stochastic modeling of flow-structure interactions using generalized polynomial chaos. Journal of Fluids Engineering. 2002;124:51. DOI: 10.1115/1.1436089 [23] Eldred M, Burkardt J. Comparison of non-intrusive polynomial chaos and stochastic collocation methods for uncertainty quantification. In: 47th AIAA Aerospace Sciences Meeting including the New Horizons Forum and Aerospace Exposition. Reston, Virigina: American Institute of Aeronautics and Astronautics; 2009. DOI: 10.2514/ 6.2009-976 [24] Mcgrattan K, Mcdermott R. Fire Dynamics Simulator User's Guide. 6th ed. 2016. DOI: 10.6028/NIST.SP.1019 [25] McGrattan K, Hostikka S, McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator Technical Reference Guide Volume 1: Mathematical Model. NIST Special Publications. 1018-1. 1; 2017. DOI: 10.6028/NIST.SP.1018 [26] Menage D, Chetehouna K, Mell W. Numerical simulations of fire spread in a Pinus pinaster needles fuel bed. Journal of Physics Conference Series. 2012;395. DOI: 10.1088/1742-6596/395/1/012011 [27] Newman JS. Experimental evaluation of fire-induced stratification. Combustion and Flame. 1984;39:33-39 [28] Tang F, Li LJ, Dong MS, Wang Q, Mei FZ, Hu LH. Characterization of buoyant flow stratification behaviors by Richardson (Froude) number in a tunnel fire with complex combination of longitudinal ventilation and ceiling extraction. Applied Thermal Engineering. 2017;110:1021-1028. DOI: 10.1016/j.applthermaleng.2016.08.224 [29] Huang DF, Li SC. An experimental investigation of stratification characteristic of fire smoke in the corridor under the effect of outdoor wind. Journal of Wind Engineering and Industrial Aerodynamics. 2018;179:173-183. DOI: 10.1016/j.jweia.2018.05.021 31 Chapter 3 Abstract interface. 1. Introduction Shifting Wildfire Trends and Twenty-first Century Rebecca Abney and Qin Ma Management Implications for the Anthropogenic climate change is projected to impact a significant proportion of ecosystems throughout the world. These shifts in climate are already impacting a diversity of wildland and urban ecosystems, and they are projected to increase wildfire frequency and severity in many regions. This projected increase is the result of the interaction of altered drought, precipitation, and temperature regimes. Understanding shifts in wildfire regimes is critical for managers at the wildlandurban interface that work to protect structures and human life. This chapter will explore how ongoing and future shifts in climate will drive alterations to natural fire regimes in the United States, with focus on implications for the wildland-urban Keywords: climate change, fire regime, urban-natural interface, wildfire infrastructure has become more damaging and costly. Fire is a global phenomenon that has historically maintained the structure and function of a range of ecosystems. Many ecosystems are adapted to periodic fire events, known as fire regimes, that describe the interval and severity of fire in a particular system. However, human influences in the twentieth century have changed the frequency and severity of wildfire in many forested ecosystems and understanding these shifts of fire regimes has been a major topic of investigation for the past several decades. This research has elucidated the numerous, complex, and interactive environmental factors driving shifts in wildfire regimes. Annually, 450 mHa of the Earth surface is burned due to wildfire [1], and the severity of wildland fires across the US has increased since the 1980s [2]. This is important because as the size, severity, and frequency of fires have changed, their influence on human The wildland-urban interface (WUI) is the boundary where human civilization and unmanaged lands meet. Currently, this interface occupies over 770,000 km2 in the US, and increases in area classified as WUI are driven by ongoing development that pushes urban environments further into wildland areas [3]. Increasing development into the WUI puts increasing numbers of structures, mainly residential homes, and human lives at risk to damage or loss via wildfire. Further, the Wildland Urban Interface in the #### Chapter 3 Safety. 2018;170:20-30. DOI: 10.1016/j. Fire Safety and Management Awareness generalized polynomial chaos. Journal of Fluids Engineering. 2002;124:51. DOI: [23] Eldred M, Burkardt J. Comparison of non-intrusive polynomial chaos and stochastic collocation methods for uncertainty quantification. In: 47th AIAA Aerospace Sciences Meeting including the New Horizons Forum and Aerospace Exposition. Reston, Virigina: American Institute of Aeronautics and Astronautics; 2009. DOI: 10.2514/ [24] Mcgrattan K, Mcdermott R. Fire Dynamics Simulator User's Guide. 6th ed. 2016. DOI: 10.6028/NIST.SP.1019 McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator Technical Reference Guide Volume 1: Mathematical Model. NIST Special Publications. 1018-1. 1; 2017. DOI: [26] Menage D, Chetehouna K, Mell W. Numerical simulations of fire spread in a Pinus pinaster needles fuel bed. Journal of Physics Conference Series. 2012;395. DOI: 10.1088/1742-6596/395/1/012011 evaluation of fire-induced stratification. Combustion and Flame. 1984;39:33-39 [28] Tang F, Li LJ, Dong MS, Wang Q, Mei FZ, Hu LH. Characterization of buoyant flow stratification behaviors by Richardson (Froude) number in a tunnel fire with complex combination of longitudinal ventilation and ceiling extraction. Applied Thermal Engineering. 2017;110:1021-1028. DOI: 10.1016/j.applthermaleng.2016.08.224 [29] Huang DF, Li SC. An experimental characteristic of fire smoke in the corridor under the effect of outdoor wind. Journal of Wind Engineering and Industrial Aerodynamics. 2018;179:173-183. DOI: investigation of stratification 10.1016/j.jweia.2018.05.021 [27] Newman JS. Experimental [25] McGrattan K, Hostikka S, 10.6028/NIST.SP.1018 10.1115/1.1436089 6.2009-976 Chetehouna K, de Izarra C, Nait-Said R, Zidani F. BLEVE fireball modeling using fire dynamics simulator (FDS) in an Algerian gas industry. Journal of Loss Prevention in the Process Industries. 2018;54. DOI: 10.1016/j.jlp.2018.02.010 [16] Magnognou B, Garo JP, Coudour B, Wang HY. Risk analysis of unburnt gas ignition in an exhaust system connected to a confined and mechanically ventilated enclosure fire. Fire Safety Journal. 2017;91:291-302. DOI: 10.1016/ [17] Saltelli A, Ratto M, Tarantola S, Campolongo F. Sensitivity Analysis Practice: A Guide to Scientific Models. 2006. DOI: 10.1016/j.ress.2005.11.014 [18] Sobol IM. Sensitivity analysis for nonlinear mathematical models. Mathematical Modeling and Computation. 1993;1:407-414. DOI: 10.18287/0134-2452-2015-39-4-459-461 [20] Chaos M. Application of sensitivity analyses to condensed-phase pyrolysis modeling. Fire Safety Journal. 2013;61: 254-264. DOI: 10.1016/j.firesaf.2013. [21] Yi L, Gao Y, Niu JL, Yang SJ. Study on effect of wind on natural smoke exhaust of enclosure fire with a twolayer zone model. Journal of Wind Aerodynamics. 2013;119:28-38. DOI: Karniadakis GE. Stochastic modeling of flow-structure interactions using Engineering and Industrial 10.1016/j.jweia.2013.05.005 [22] Xiu D, Lucor D, Su C-H, [19] Crestaux T, Le Maître O, Martinez JM. Polynomial chaos expansion for sensitivity analysis. Reliability Engineering and System Safety. 2009;94:1161-1172. DOI: 10.1016/j.ress.2008.10.008 09.016 30 j.firesaf.2017.03.036 ress.2017.10.007 [15] Sellami I, Manescau B, ## Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface in the Twenty-first Century Rebecca Abney and Qin Ma ## Abstract Anthropogenic climate change is projected to impact a significant proportion of ecosystems throughout the world. These shifts in climate are already impacting a diversity of wildland and urban ecosystems, and they are projected to increase wildfire frequency and severity in many regions. This projected increase is the result of the interaction of altered drought, precipitation, and temperature regimes. Understanding shifts in wildfire regimes is critical for managers at the wildlandurban interface that work to protect structures and human life. This chapter will explore how ongoing and future shifts in climate will drive alterations to natural fire regimes in the United States, with focus on implications for the wildland-urban interface. Keywords: climate change, fire regime, urban-natural interface, wildfire #### 1. Introduction Fire is a global phenomenon that has historically maintained the structure and function of a range of ecosystems. Many ecosystems are adapted to periodic fire events, known as fire regimes, that describe the interval and severity of fire in a particular system. However, human influences in the twentieth century have changed the frequency and severity of wildfire in many forested ecosystems and understanding these shifts of fire regimes has been a major topic of investigation for the past several decades. This research has elucidated the numerous, complex, and interactive environmental factors driving shifts in wildfire regimes. Annually, 450 mHa of the Earth surface is burned due to wildfire [1], and the severity of wildland fires across the US has increased since the 1980s [2]. This is important because as the size, severity, and frequency of fires have changed, their influence on human infrastructure has become more damaging and costly. The wildland-urban interface (WUI) is the boundary where human civilization and unmanaged lands meet. Currently, this interface occupies over 770,000 km2 in the US, and increases in area classified as WUI are driven by ongoing development that pushes urban environments further into wildland areas [3]. Increasing development into the WUI puts increasing numbers of structures, mainly residential homes, and human lives at risk to damage or loss via wildfire. Further, the infrastructure required by the WUI presents an additional source of ignitions in areas that are primed to burn. While trees exhibit traits of fire resistance [4, 5], houses, in particular older structures, burn with greater intensity and speed. For example, the 2018 Camp Fire in the Sierra Nevada of California burned quickly through the town of Paradise while leaving many standing trees scorched but not completely burnt. While this fire had many complex causes [6], the quick spread of the fire through the town was a reason that escape was made difficult despite a populous aware and prepared for the danger. While these changes in fire regimes have exacerbated the damage in WUI, anthropogenic climate change is expected to intensify the risk by fire to WUIs. Across the US, climate change in the next century is projected to drive increases in wildfire severity in some areas, and increased wildfire incidence in other areas [7]. Shifts in wildfire patterns will be driven by shifts in precipitation timing and amounts, vegetation, temperature regimes, and drought conditions [8–11]. While changing climate patterns have been reasonably well characterized, wildfire regimes are more complex to predict due to the interconnected nature of the drivers and heterogeneous nature of ignition sources. It is critical to understand and provide more accurate predictions for shifts in wildfire frequency and severity, due to the loss of life, economic damage, related catastrophic environmental events, such as flooding or water quality damage. This is particularly important as human development into the wildland areas, which are more prone to wildfires, has increased significantly over the past half century. #### 2. Shifting wildfire regimes Fire regimes integrate the tendency of vegetation to burn and the climate conditions that promote fire in a metric that describes the spatial and temporal nature of fire in a particular region. While there are several ways to calculate these metrics [12] a general calculation includes a measure of how frequently a fire occurs at a location (i.e., the average fire return interval) and the effect that fire has on vegetation (i.e., the severity of the fire). Variability in fire regimes is driven by differences in elevation, vegetation life history, drought and precipitation patterns, land-use, among other ecosystem-specific parameters [13, 14]. Many animal and plant species have co-evolved with fire and are adapted to specific fire regimes [15]. Some densergrowing vegetation species are adapted to higher severity and stand-replacing burns, such as in the Northern Rockies, while other species are more adapted to lower and more moderate severity burns, such as in the southern Sierra Nevada. The inherent complexity and spatial heterogeneity of fire regimes make it difficult to make general recommendations for fire management [15]. However, the implications of an expanding WUI and increasing trends of fire activity indicate a clear problem for fire management. This is compounded by the possibility that fire regimes may shift over time in response to anthropogenic driven changes in management, vegetation composition and density, and climate [16, 17]. #### 2.1 History of fire regimes in the US Historically, fire regimes were mostly driven by an ecosystem's vegetation, climate conditions, and human activities, which varied both spatially and temporally over the US. In the Northern Rocky Mountains, stand replacing fires are typical in pine forests of the region [18, 19]. Fires in this ecosystem occur at relatively low frequency (longer return intervals), but when they do occur, they can burn large areas of forest ecosystems at high severity, e.g., the Yellowstone fire in 1988 [20–22]. 33 Figure 1. All photos are © R. Abney. Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… In contrast, low-intensity fires occurred more frequently in the southwestern forests of New Mexico and Arizona, due to the dry and warm semi-arid climate and tree species that exhibited resistance to fires (e.g., Pinus ponderosa). Much of the southeast was historically occupied by longleaf pine (Pinus palustris, Figure 1A), which thrives in high frequency, low burn severity fire regimes [23]. Pre-European settlement, longleaf pine forests were managed by Native American populations, and post-settlement there was a significant decline in the land area of these forests [24]. Currently, they are managed via frequent prescribed fire and are often grown to produce pine straw [25]. While wildfires have been major drivers of the American landscape, natural ecosystems are continually adapting to fire regimes over time in response to shifts in vegetation, management, and climate. However, human activities, management, and climate change are also driving the interaction between fire The shift in fire regimes in the Sierra Nevada is an example of the interactive effects of human management and climate change. Prior to Euro-American settlement, natural lighting strikes and fire activities by Native Americans were the main causes of fire ignitions in the Sierra Nevada [26]. Forests were burned with mixed-severity fires that included both light to moderate burning of understory and crown fires at the interval of a decade or two. The small trees and ground fuels were killed and cleaned in fires periodically, leaving patches of large, mature trees that are more resistant to wildfires due to thick bark that is hard to burn, preventing fire from spreading to the canopy [4, 5]. However, a combination of human influences changed the structure of these forests and made them more susceptible to frequent fires that spread through canopies. Early twentieth century logging practices preferentially selected for these larger trees, opening up space for denser thickets of small trees to colonize, leading to increases in forest density [27]. This change in structure was reinforced by widespread suppression of fires that historically cleared out undergrowth. Since the early twentieth century, fire suppression as a forest management technique was widely adopted after several large and severe wildfires Fire severity is in part controlled by the density of the fuels and fire return interval. In photo A, loblolly pine (Pinus palustris) plantation in Georgia is managed for pulpwood production with prescribed fire approximately every 3 years. This low severity, high frequency fire regime maintains an open canopy and lowdensity fuels. In photo B, a conifer forest in Yosemite National Park one-year post recovery after the Rim Fire. DOI: http://dx.doi.org/10.5772/intechopen.93245 and vegetation over the past several centuries. #### Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… DOI: http://dx.doi.org/10.5772/intechopen.93245 In contrast, low-intensity fires occurred more frequently in the southwestern forests of New Mexico and Arizona, due to the dry and warm semi-arid climate and tree species that exhibited resistance to fires (e.g., Pinus ponderosa). Much of the southeast was historically occupied by longleaf pine (Pinus palustris, Figure 1A), which thrives in high frequency, low burn severity fire regimes [23]. Pre-European settlement, longleaf pine forests were managed by Native American populations, and post-settlement there was a significant decline in the land area of these forests [24]. Currently, they are managed via frequent prescribed fire and are often grown to produce pine straw [25]. While wildfires have been major drivers of the American landscape, natural ecosystems are continually adapting to fire regimes over time in response to shifts in vegetation, management, and climate. However, human activities, management, and climate change are also driving the interaction between fire and vegetation over the past several centuries. The shift in fire regimes in the Sierra Nevada is an example of the interactive effects of human management and climate change. Prior to Euro-American settlement, natural lighting strikes and fire activities by Native Americans were the main causes of fire ignitions in the Sierra Nevada [26]. Forests were burned with mixed-severity fires that included both light to moderate burning of understory and crown fires at the interval of a decade or two. The small trees and ground fuels were killed and cleaned in fires periodically, leaving patches of large, mature trees that are more resistant to wildfires due to thick bark that is hard to burn, preventing fire from spreading to the canopy [4, 5]. However, a combination of human influences changed the structure of these forests and made them more susceptible to frequent fires that spread through canopies. Early twentieth century logging practices preferentially selected for these larger trees, opening up space for denser thickets of small trees to colonize, leading to increases in forest density [27]. This change in structure was reinforced by widespread suppression of fires that historically cleared out undergrowth. Since the early twentieth century, fire suppression as a forest management technique was widely adopted after several large and severe wildfires #### Figure 1. Fire Safety and Management Awareness over the past half century. 2. Shifting wildfire regimes 2.1 History of fire regimes in the US populous aware and prepared for the danger. infrastructure required by the WUI presents an additional source of ignitions in areas that are primed to burn. While trees exhibit traits of fire resistance [4, 5], houses, in particular older structures, burn with greater intensity and speed. For example, the 2018 Camp Fire in the Sierra Nevada of California burned quickly through the town of Paradise while leaving many standing trees scorched but not completely burnt. While this fire had many complex causes [6], the quick spread of the fire through the town was a reason that escape was made difficult despite a While these changes in fire regimes have exacerbated the damage in WUI, anthropogenic climate change is expected to intensify the risk by fire to WUIs. Across the US, climate change in the next century is projected to drive increases in wildfire severity in some areas, and increased wildfire incidence in other areas [7]. Shifts in wildfire patterns will be driven by shifts in precipitation timing and amounts, vegetation, temperature regimes, and drought conditions [8–11]. While changing climate patterns have been reasonably well characterized, wildfire regimes are more complex to predict due to the interconnected nature of the drivers and heterogeneous nature of ignition sources. It is critical to understand and provide more accurate predictions for shifts in wildfire frequency and severity, due to the loss of life, economic damage, related catastrophic environmental events, such as flooding or water quality damage. This is particularly important as human development into the wildland areas, which are more prone to wildfires, has increased significantly Fire regimes integrate the tendency of vegetation to burn and the climate conditions that promote fire in a metric that describes the spatial and temporal nature of fire in a particular region. While there are several ways to calculate these metrics [12] a general calculation includes a measure of how frequently a fire occurs at a location (i.e., the average fire return interval) and the effect that fire has on vegetation (i.e., the severity of the fire). Variability in fire regimes is driven by differences in elevation, vegetation life history, drought and precipitation patterns, land-use, among other ecosystem-specific parameters [13, 14]. Many animal and plant species have co-evolved with fire and are adapted to specific fire regimes [15]. Some densergrowing vegetation species are adapted to higher severity and stand-replacing burns, such as in the Northern Rockies, while other species are more adapted to lower and more moderate severity burns, such as in the southern Sierra Nevada. agement, vegetation composition and density, and climate [16, 17]. The inherent complexity and spatial heterogeneity of fire regimes make it difficult to make general recommendations for fire management [15]. However, the implications of an expanding WUI and increasing trends of fire activity indicate a clear problem for fire management. This is compounded by the possibility that fire regimes may shift over time in response to anthropogenic driven changes in man- Historically, fire regimes were mostly driven by an ecosystem's vegetation, climate conditions, and human activities, which varied both spatially and temporally over the US. In the Northern Rocky Mountains, stand replacing fires are typical in pine forests of the region [18, 19]. Fires in this ecosystem occur at relatively low frequency (longer return intervals), but when they do occur, they can burn large areas of forest ecosystems at high severity, e.g., the Yellowstone fire in 1988 [20–22]. 32 Fire severity is in part controlled by the density of the fuels and fire return interval. In photo A, loblolly pine (Pinus palustris) plantation in Georgia is managed for pulpwood production with prescribed fire approximately every 3 years. This low severity, high frequency fire regime maintains an open canopy and lowdensity fuels. In photo B, a conifer forest in Yosemite National Park one-year post recovery after the Rim Fire. All photos are © R. Abney. in the Northern Rockies that killed many and destroyed a number of settlements. The fire suppression efforts were successful in excluding low-severity fires, and this management strategy reduced the fire frequency to the lowest frequency measured in the past 3000 years [28]. Consequently, the accompanying densification of forests due to the fire deficit has contributed to increasing numbers of devastating fires in late twentieth and twenty-first centuries [29]. This shift in fire regimes is the result of combined factors including (1) the reduction of regular fire usage, which were regularly conducted by Native Americans to reduce fuel loads and to encourage culturally important vegetation [30]; (2) legacy of decades of fire-suppression that densified undergrowth which lead to increased spread of fire; (3) removal of large trees, which are resilient to low-to-medium fires, due to industrialized timber logging; (4) the disappearing of gaps among trees, which could have stopped fire from spreading, but were filled with smaller and denser trees that can easily act as continuous fuel sources and (5) species change from those with fire adverse traits, to shade-tolerant ones [31]. The current fire regime that includes more high-severity, large fire size, is a significant challenge to forest managers and is a critical risk to the safety of human life and development in the WUI. #### 2.2 Drivers of wildfire change Drivers of wildfire include three main categories: regional climate, fuel availability and condition, and ignition sources. In areas of low fuel density, sources of ignition drive fire occurrence; however, in higher population density areas, such as the WUI, fuel availability drives fire occurrence [32]. Climate influences fire occurrence by the timing and amount of precipitation, temperature, and wind speed. Wildfire season starts when all these climate features reach their thresholds. The intensity of drought and strength of wind as well as the length of wildfire season is highly related to the severity and risk of wildfires. Westerling et al. [17] found that an extended fire season, resulting from earlier spring warming and extended drought in late fall, increased the fire frequency and severity in the Western US. This trend is predicted to continue as climate gets warmer and drier with ongoing climate change [7]. In the eastern US, precipitation and temperature patterns form a different climate, and thus different fire seasons than the western US. Southwestern forests are influenced by late-summer precipitation stemming from the North American monsoon that end fire-season earlier in the year. The pacific north-west and the Northern Rockies are routinely colder and wetter, thus interannual fire season lengths are short in general. The available fuel load in part determines the extent of wildfire, including what and how much can be burned. In areas with limited fuel loads, such as the shrubland and grassland in Southwestern US, fires can occur frequently but are usually low-severity burns. High severity burns often occur in forests with large and dense biomass, which can provide plentiful fuel sources for wildfires. The spatial continuity of fuels also plays a critical role in shifts in fire regimes. The combination of large trees and clearings in forest floor vegetation in historical frequent-fire Western forests constrained the spread of crown fires. Examples of this are found in ponderosa or giant sequoia groves. However, effective fire suppression until the 1980s has reduced the number of surface fires that would have removed the ground and understory fuels periodically. Small trees and undergrowth filled the gaps between trunks and created continuous fuels that could carry flames to tree crowns, which has in part lead to higher severity and larger fires in the Western US that are currently observed [33] (Figure 2). Thus, forest and fire management can change fire regimes by changing the quantity and structure of fuels. 35 influence [28]. Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… Ignitions are a critical factor of wildfire regimes. Before the European settlement, lightning and Native American activities were the sources of ignition. As populations and permanent infrastructure expanded in the past century, sources of ignitions diversified, particularly in the WUI. While lightning is still an ignition source of large, severe wildfires in areas of lower population density such as in boreal forests and at higher latitudes [29], more fires are ignited by Anthropogenic sources, particularly as the WUI expands, such as sparks from power lines [34], The number of fires and land area burned in wildlands. Data are from the National Interagency Fire Center [2]. The number of fires has remained relatively constant since the 1980s, but the land area burned has The interactions among the three factors can change fire regimes in a positive feedback cycle. In areas with low population and human activities, sources of fire ignition increase fire occurrence, but in areas with high population density and frequent human activities, fuel availability drives the fire regime. In the meanwhile, shifts in climate can either increase or decrease the probability of fire occurrences in Prior to Native American settlement of North America, wildfires were unmanaged, and their severity and frequency were a result of the available fuel load and local climatic factors, namely precipitation, temperature, and drought conditions [37, 38]. North America was settled approximately 14,000 years ago [39], and there is considerable evidence for management of landscapes by Native Americans [40]. The exact magnitude of Native American burning is difficult to determine, due to methodological limitations in reconstructing historic fire frequencies [41], but the available evidence suggests that Native Americans utilized low severity burns in order to maintain prairie habitats and encourage growth of vegetation for cultural usage [40, 42]. The reconstructed fire record of the western US suggests that much of the pre-European settlement wildfire regime was primarily dictated by large-scale climate patterns, rather than via human Around the turn of the twenty-first century, policies were introduced to encourage fire suppression, mainly wildland firefighting, in part as a response to fires in the Northern Rockies in 1910 and as a means to protect timber resources and human accidental flares from camping fires [35], and deliberate arson [36]. increased, indicating that fires are becoming larger and more catastrophic. addition to the other two drivers. Figure 2. 2.3 Shifts in wildfire management DOI: http://dx.doi.org/10.5772/intechopen.93245 Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… DOI: http://dx.doi.org/10.5772/intechopen.93245 #### Figure 2. Fire Safety and Management Awareness the safety of human life and development in the WUI. 2.2 Drivers of wildfire change season lengths are short in general. in the Northern Rockies that killed many and destroyed a number of settlements. The fire suppression efforts were successful in excluding low-severity fires, and this management strategy reduced the fire frequency to the lowest frequency measured in the past 3000 years [28]. Consequently, the accompanying densification of forests due to the fire deficit has contributed to increasing numbers of devastating fires in late twentieth and twenty-first centuries [29]. This shift in fire regimes is the result of combined factors including (1) the reduction of regular fire usage, which were regularly conducted by Native Americans to reduce fuel loads and to encourage culturally important vegetation [30]; (2) legacy of decades of fire-suppression that densified undergrowth which lead to increased spread of fire; (3) removal of large trees, which are resilient to low-to-medium fires, due to industrialized timber logging; (4) the disappearing of gaps among trees, which could have stopped fire from spreading, but were filled with smaller and denser trees that can easily act as continuous fuel sources and (5) species change from those with fire adverse traits, to shade-tolerant ones [31]. The current fire regime that includes more high-severity, large fire size, is a significant challenge to forest managers and is a critical risk to Drivers of wildfire include three main categories: regional climate, fuel availability and condition, and ignition sources. In areas of low fuel density, sources of ignition drive fire occurrence; however, in higher population density areas, such as the WUI, fuel availability drives fire occurrence [32]. Climate influences fire occurrence by the timing and amount of precipitation, temperature, and wind speed. Wildfire season starts when all these climate features reach their thresholds. The intensity of drought and strength of wind as well as the length of wildfire season is highly related to the severity and risk of wildfires. Westerling et al. [17] found that an extended fire season, resulting from earlier spring warming and extended drought in late fall, increased the fire frequency and severity in the Western US. This trend is predicted to continue as climate gets warmer and drier with ongoing climate change [7]. In the eastern US, precipitation and temperature patterns form a different climate, and thus different fire seasons than the western US. Southwestern forests are influenced by late-summer precipitation stemming from the North American monsoon that end fire-season earlier in the year. The pacific north-west and the Northern Rockies are routinely colder and wetter, thus interannual fire The available fuel load in part determines the extent of wildfire, including what and how much can be burned. In areas with limited fuel loads, such as the shrubland and grassland in Southwestern US, fires can occur frequently but are usually low-severity burns. High severity burns often occur in forests with large and dense biomass, which can provide plentiful fuel sources for wildfires. The spatial continuity of fuels also plays a critical role in shifts in fire regimes. The combination of large trees and clearings in forest floor vegetation in historical frequent-fire Western forests constrained the spread of crown fires. Examples of this are found in ponderosa or giant sequoia groves. However, effective fire suppression until the 1980s has reduced the number of surface fires that would have removed the ground and understory fuels periodically. Small trees and undergrowth filled the gaps between trunks and created continuous fuels that could carry flames to tree crowns, which has in part lead to higher severity and larger fires in the Western US that are currently observed [33] (Figure 2). Thus, forest and fire management can change fire regimes by changing the quantity and structure of fuels. 34 The number of fires and land area burned in wildlands. Data are from the National Interagency Fire Center [2]. The number of fires has remained relatively constant since the 1980s, but the land area burned has increased, indicating that fires are becoming larger and more catastrophic. Ignitions are a critical factor of wildfire regimes. Before the European settlement, lightning and Native American activities were the sources of ignition. As populations and permanent infrastructure expanded in the past century, sources of ignitions diversified, particularly in the WUI. While lightning is still an ignition source of large, severe wildfires in areas of lower population density such as in boreal forests and at higher latitudes [29], more fires are ignited by Anthropogenic sources, particularly as the WUI expands, such as sparks from power lines [34], accidental flares from camping fires [35], and deliberate arson [36]. The interactions among the three factors can change fire regimes in a positive feedback cycle. In areas with low population and human activities, sources of fire ignition increase fire occurrence, but in areas with high population density and frequent human activities, fuel availability drives the fire regime. In the meanwhile, shifts in climate can either increase or decrease the probability of fire occurrences in addition to the other two drivers. #### 2.3 Shifts in wildfire management Prior to Native American settlement of North America, wildfires were unmanaged, and their severity and frequency were a result of the available fuel load and local climatic factors, namely precipitation, temperature, and drought conditions [37, 38]. North America was settled approximately 14,000 years ago [39], and there is considerable evidence for management of landscapes by Native Americans [40]. The exact magnitude of Native American burning is difficult to determine, due to methodological limitations in reconstructing historic fire frequencies [41], but the available evidence suggests that Native Americans utilized low severity burns in order to maintain prairie habitats and encourage growth of vegetation for cultural usage [40, 42]. The reconstructed fire record of the western US suggests that much of the pre-European settlement wildfire regime was primarily dictated by large-scale climate patterns, rather than via human influence [28]. Around the turn of the twenty-first century, policies were introduced to encourage fire suppression, mainly wildland firefighting, in part as a response to fires in the Northern Rockies in 1910 and as a means to protect timber resources and human Figure 3. Number of prescribed fires and acres burned in the United States from 1997 to 2018, data from the National Interagency Fire Center. settlements [43, 44]. These policies generally did not consider fire suppression via other management strategies (e.g., fuel load reductions, prescribed burning), which led to a significant increase in the density of American forests [43]. In the past several decades, scientific research indicated the role that fires play in natural ecosystems in shaping ecosystem dynamics, but also to prevent the large fuel loading that results in larger, more severe wildfires. Following this research and shifts in political perspectives, recent changes in legislation, namely the Healthy Forests Initiative (2002) and the Healthy Forests Restoration Act (2003) [44], have allowed for more prescribed burning (Figure 3). This rapid increase in the use of prescribed fire across the US is likely to lead to a shift back towards a more natural fire regime in some areas, although it is unlikely that the magnitude of prescribed burning would approach the extent of what would naturally occur. Prescribed burning has been widely adopted in the southeastern US, which in recent decades has led to a decrease in wildfires, with some exceptions in drought years [45]. In the western US, prescribed burning has been slower to be more widely adopted as a management strategy due to a number of factors, including the larger proportion of public lands, more restrictive legislation, and concerns about emissions and air quality [46]. Across the US, considerable public weariness of prescribed fire has also been a major barrier to its widespread use [46], due to concerns about control of the burns and air quality. #### 2.4 Predictions for future wildfire regimes Projections for future wildfire regimes indicate that some areas of the US will experience larger and more severe wildfires, while other areas will experience fewer and less severe wildfires. The accuracy of these projections will in part depend upon management techniques within fire-prone ecosystems, including the use of prescribed burning vs. fire suppression [16]. In their recent study, Parks, Miller [16] project significant decreases in wildfire severity in the western US, which they attribute to changes in fuel loads into the twenty-first century and water deficit conditions. In the southeastern US, projections indicate a slight increase in area burned, with considerable variability across different states [47]. 37 Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… The major concerns of wildfire in the WUI are the risk to human life, structures, and economic productivity. The WUI comprises 9% of the land in the US, which equates to 39% of all housing units [48]. Prior development increased the proportion of land classified as a WUI from 1970 to 2000 by 52%, with future projections for One major consideration for management of wildfire risk at the WUI is understanding the drivers of shifts in wildfire regimes into the future. Some modeling work has predicted that shifts in fire regimes into the future will be more significant for wildfire occurrence at the WUI than expansion of WUI development [50]. However, with increasing areas classified as WUI, there are also increasing ignition sources for wildfires and developed lands that could suffer wildfire damage [3]. Some modeling work has shown that whether a residence has fire proofing, and the density of surrounding homes and vegetation all interact to control the severity and size of wildfire [51]. 3.1 Current and shifting attitudes towards fire in the wildland-urban interface considerable research attention, because frequently public perception of the use of wildfire management techniques prevents their use [52–54]. Some of the major concerns are related to the cost of implementation of the management technique and direct impacts during implementation, such as decreased air quality during prescribed fire, and drawbacks of particular fire management techniques, including costs [53, 54]. Public attitudes towards wildfire management at the WUI also depend upon local factors, including previous wildfire management strategies employed, trust in local agencies responsible for managing wildfire risk, and individual atti- 3.2 Public awareness of shifting climates at the wildland urban interface 4. Management of wildfire in the wildland-urban interface 4.1 Current strategies for managing wildfire at the wildland-urban interface The main historic and current strategy to reduce the risk of wildfire has been fuel reduction [3, 54]. In wildlands, prescribed fire, allowing natural fires to burn within designated boundaries, and mechanical treatments, such as thinning or mastication, are the main strategies that have been successfully used to reduce wildfire frequency and severity [60]. There is a need to develop or re-develop the natural fire regime, or shift towards a more frequent, lower intensity fire regime, As development has continued into the wildland-urban interface over the past several centuries, wildfire severity has increased [40]. Recent research has indicated that some populations are aware that future shifts in climate may lead to increased risk of wildfire and related property damage [57]. However, public perceptions of climate change have not significantly shifted in the past several decades, except along some partisan divides [58]. Regardless of public awareness of shifting wildfire risk into the future, areas of increased risk are facing increased insurance premiums tudes towards the management techniques [53, 55, 56]. and rates, as they already have in California [59]. particularly in the Western US [40]. Understanding attitudes concerning wildfire management at the WUI has drawn DOI: http://dx.doi.org/10.5772/intechopen.93245 ongoing increases in WUI lands [49]. 3. Wildfire in the wildland-urban interface Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… DOI: http://dx.doi.org/10.5772/intechopen.93245 #### 3. Wildfire in the wildland-urban interface Fire Safety and Management Awareness settlements [43, 44]. These policies generally did not consider fire suppression via other management strategies (e.g., fuel load reductions, prescribed burning), which Number of prescribed fires and acres burned in the United States from 1997 to 2018, data from the National In the past several decades, scientific research indicated the role that fires play in natural ecosystems in shaping ecosystem dynamics, but also to prevent the large fuel loading that results in larger, more severe wildfires. Following this research and shifts in political perspectives, recent changes in legislation, namely the Healthy Forests Initiative (2002) and the Healthy Forests Restoration Act (2003) [44], have allowed for more prescribed burning (Figure 3). This rapid increase in the use of prescribed fire across the US is likely to lead to a shift back towards a more natural fire regime in some areas, although it is unlikely that the magnitude of prescribed burning would approach the extent of what would Prescribed burning has been widely adopted in the southeastern US, which in recent decades has led to a decrease in wildfires, with some exceptions in drought years [45]. In the western US, prescribed burning has been slower to be more widely adopted as a management strategy due to a number of factors, including the larger proportion of public lands, more restrictive legislation, and concerns about emissions and air quality [46]. Across the US, considerable public weariness of prescribed fire has also been a major barrier to its widespread use [46], due to concerns Projections for future wildfire regimes indicate that some areas of the US will experience larger and more severe wildfires, while other areas will experience fewer and less severe wildfires. The accuracy of these projections will in part depend upon management techniques within fire-prone ecosystems, including the use of prescribed burning vs. fire suppression [16]. In their recent study, Parks, Miller [16] project significant decreases in wildfire severity in the western US, which they attribute to changes in fuel loads into the twenty-first century and water deficit conditions. In the southeastern US, projections indicate a slight increase in area burned, led to a significant increase in the density of American forests [43]. 36 naturally occur. Figure 3. Interagency Fire Center. about control of the burns and air quality. 2.4 Predictions for future wildfire regimes with considerable variability across different states [47]. The major concerns of wildfire in the WUI are the risk to human life, structures, and economic productivity. The WUI comprises 9% of the land in the US, which equates to 39% of all housing units [48]. Prior development increased the proportion of land classified as a WUI from 1970 to 2000 by 52%, with future projections for ongoing increases in WUI lands [49]. One major consideration for management of wildfire risk at the WUI is understanding the drivers of shifts in wildfire regimes into the future. Some modeling work has predicted that shifts in fire regimes into the future will be more significant for wildfire occurrence at the WUI than expansion of WUI development [50]. However, with increasing areas classified as WUI, there are also increasing ignition sources for wildfires and developed lands that could suffer wildfire damage [3]. Some modeling work has shown that whether a residence has fire proofing, and the density of surrounding homes and vegetation all interact to control the severity and size of wildfire [51]. #### 3.1 Current and shifting attitudes towards fire in the wildland-urban interface Understanding attitudes concerning wildfire management at the WUI has drawn considerable research attention, because frequently public perception of the use of wildfire management techniques prevents their use [52–54]. Some of the major concerns are related to the cost of implementation of the management technique and direct impacts during implementation, such as decreased air quality during prescribed fire, and drawbacks of particular fire management techniques, including costs [53, 54]. Public attitudes towards wildfire management at the WUI also depend upon local factors, including previous wildfire management strategies employed, trust in local agencies responsible for managing wildfire risk, and individual attitudes towards the management techniques [53, 55, 56]. #### 3.2 Public awareness of shifting climates at the wildland urban interface As development has continued into the wildland-urban interface over the past several centuries, wildfire severity has increased [40]. Recent research has indicated that some populations are aware that future shifts in climate may lead to increased risk of wildfire and related property damage [57]. However, public perceptions of climate change have not significantly shifted in the past several decades, except along some partisan divides [58]. Regardless of public awareness of shifting wildfire risk into the future, areas of increased risk are facing increased insurance premiums and rates, as they already have in California [59]. #### 4. Management of wildfire in the wildland-urban interface #### 4.1 Current strategies for managing wildfire at the wildland-urban interface The main historic and current strategy to reduce the risk of wildfire has been fuel reduction [3, 54]. In wildlands, prescribed fire, allowing natural fires to burn within designated boundaries, and mechanical treatments, such as thinning or mastication, are the main strategies that have been successfully used to reduce wildfire frequency and severity [60]. There is a need to develop or re-develop the natural fire regime, or shift towards a more frequent, lower intensity fire regime, particularly in the Western US [40]. Land managers of ecosystems that are highly prone to wildfire at the WUI will likely need to undertake a proactive management approach to protect human safety and infrastructure in the WUI [40]. A commonly utilized strategy at the WUI is the establishment of a "defensible space" around residences and other properties, which reduces vegetation and other burn hazards adjacent and up to 30 m away from buildings [61]. Buildings can also be constructed of combustion-resistant materials, although this strategy is more effective when combined with defensible space [62]. #### 4.2 Recommendations for strategies in consideration of future climate and fire regimes While many strategies have been identified to manage forests at the wildlandurban interface, they have not been widely adopted due to a combination of factors, including lack of funding and political willpower [63]. Current research has indicated the effectiveness of utilizing prescribed fire to reduce the frequency and severity [45]. Expanded and more frequent use of prescribed fire and other fuel reduction techniques in the WUI can serve to protect infrastructure from more catastrophic wildfire and act to re-establish a historic wildfire regime. One of the major barriers to increasing use of fuel reduction management strategies is public perception of both the use of these techniques and the increased risk of wildfire with ongoing climate change. Future management strategies should continue to include strategies for managing public perception to increase acceptance and participation in fuel management at the WUI and to increase understanding of the diverse factors involved in managing forests for both prescribed fire and wildfire events [64]. Additionally, these strategies should continue to focus on informing the public about the efficacy of defensible spaces and improve development planning to ensure greater accessibility, improved use of defensible space, and better building design [61]. #### 4.3 Future research and ongoing uncertainty There is considerable current and ongoing research focused on enhancing fire condition predictors and managing strategies related to reducing the severity and frequency of wildfires [16, 17, 65, 66]. Ongoing research in refining future climate predictions will generate considerably more certainty to predictions for future fire regimes. However, work in the area should focus more on the dynamics of wildfire at the WUI due to the critical resources that are at risk in those areas. Many of the obstacles to implanting these management strategies are political in nature, with responsibility falling to local governments operating under limited funding and variable community support [63]. Some recent research has indicated that local differences in legal liability for prescribed burning lead to significant differences in the amount of land burned via prescribed fires [67]. While features of landscapes that make them prone to wildfire have been reasonably well-described, future research on mitigating the effects of wildfire in the WUI should consider the human dimension to management decision making [46]. Historically, human management has driven much of the increase in wildfire severity, and into the future, there will be a need for management strategies that reconcile natural fire regimes with protection of human life and property at the WUI. #### 5. Conclusions Modern fire regimes are largely driven by anthropogenic activities and widely differ from pre-European and pre-Native American wildfire regimes. In the coming 39 Author details Rebecca Abney1 \* and Qin Ma2 2 Mississippi State University, Mississippi State, MS, USA \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, 1 University of Georgia, Athens, GA, USA provided the original work is properly cited. Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface…* decades and century, projected climate shifts will drive corresponding shifts in wildfire occurrence and severity, with differing projections for different regions of the US. Development in the WUI needs to be informed for how to manage local shifts in wildfire regimes to mitigate the impacts of severe wildfire, and some of the ability of an area to respond is related to public perception of the risks of wildfire. The authors would like to thank and acknowledge Joseph Crockett for comments DOI: http://dx.doi.org/10.5772/intechopen.93245 on earlier versions of this book chapter. The authors declare no conflict of interest. Acknowledgements Conflict of interest Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… DOI: http://dx.doi.org/10.5772/intechopen.93245 decades and century, projected climate shifts will drive corresponding shifts in wildfire occurrence and severity, with differing projections for different regions of the US. Development in the WUI needs to be informed for how to manage local shifts in wildfire regimes to mitigate the impacts of severe wildfire, and some of the ability of an area to respond is related to public perception of the risks of wildfire. #### Acknowledgements Fire Safety and Management Awareness regimes Land managers of ecosystems that are highly prone to wildfire at the WUI will likely need to undertake a proactive management approach to protect human safety and infrastructure in the WUI [40]. A commonly utilized strategy at the WUI is the establishment of a "defensible space" around residences and other properties, which reduces vegetation and other burn hazards adjacent and up to 30 m away from buildings [61]. Buildings can also be constructed of combustion-resistant materials, although this strategy is more effective when combined with defensible space [62]. 4.2 Recommendations for strategies in consideration of future climate and fire While many strategies have been identified to manage forests at the wildlandurban interface, they have not been widely adopted due to a combination of factors, including lack of funding and political willpower [63]. Current research has indicated the effectiveness of utilizing prescribed fire to reduce the frequency and severity [45]. Expanded and more frequent use of prescribed fire and other fuel reduction techniques in the WUI can serve to protect infrastructure from more One of the major barriers to increasing use of fuel reduction management strategies is public perception of both the use of these techniques and the increased risk of wildfire with ongoing climate change. Future management strategies should continue to include strategies for managing public perception to increase acceptance and participation in fuel management at the WUI and to increase understanding of the diverse factors involved in managing forests for both prescribed fire and wildfire events [64]. Additionally, these strategies should continue to focus on informing the public about the efficacy of defensible spaces and improve development planning to ensure greater accessibility, improved use of defensible space, and better building design [61]. There is considerable current and ongoing research focused on enhancing fire condition predictors and managing strategies related to reducing the severity and frequency of wildfires [16, 17, 65, 66]. Ongoing research in refining future climate predictions will generate considerably more certainty to predictions for future fire regimes. However, work in the area should focus more on the dynamics of wildfire Many of the obstacles to implanting these management strategies are political in nature, with responsibility falling to local governments operating under limited funding and variable community support [63]. Some recent research has indicated that local differences in legal liability for prescribed burning lead to significant differences in the amount of land burned via prescribed fires [67]. While features of landscapes that make them prone to wildfire have been reasonably well-described, future research on mitigating the effects of wildfire in the WUI should consider the human dimension to management decision making [46]. Historically, human management has driven much of the increase in wildfire severity, and into the future, there will be a need for management strategies that reconcile natural fire regimes Modern fire regimes are largely driven by anthropogenic activities and widely differ from pre-European and pre-Native American wildfire regimes. In the coming catastrophic wildfire and act to re-establish a historic wildfire regime. at the WUI due to the critical resources that are at risk in those areas. with protection of human life and property at the WUI. 4.3 Future research and ongoing uncertainty 38 5. Conclusions The authors would like to thank and acknowledge Joseph Crockett for comments on earlier versions of this book chapter. ### Conflict of interest The authors declare no conflict of interest. ## Author details Rebecca Abney1 \* and Qin Ma2 1 University of Georgia, Athens, GA, USA 2 Mississippi State University, Mississippi State, MS, USA \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. #### References* [1] Randerson JT et al. Global burned area and biomass burning emissions from small fires. Journal of Geophysical Research – Biogeosciences. 2012;117:G04012. DOI: 10.1029/2012JG002128 [2] National Interagency Fire Center. 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Influence of pine straw harvesting, prescribed fire, and fertilization on a Louisiana longleaf pine site. Southern Journal of Applied Forestry. 2009;33(3):115-120 [26] Pyne S, Vale TR. Fire, native peoples, and the natural landscape. Restoration Ecology: Book Review. 2003;11(2):257-259 [27] Naficy C et al. Interactive effects of historical logging and fire exclusion on ponderosa pine forest structure in the northern Rockies. Ecological Applications. 2010;20(7):1851-1864 [28] Marlon JR et al. Long-term perspective on wildfires in the western USA. Proceedings of the National Academy of Sciences. 2012;109(9):E535-E543 [29] National Interagency Fire Center. Prescribed Fires and Acres by Agency. 2018. Available from: https://www.nifc. gov/fireInfo/fireInfo\_statistics.html [cited 28 August 2018] [30] Stewart OC. Forgotten Fires: Native Americans and the Transient Wilderness. Norman, OK: University of Oklahoma Press; 2002 [31] Earles JM, North MP, Hurteau MD. Wildfire and drought dynamics destabilize carbon stores of fire-suppressed forests. Ecological Applications. 2014;24(4):732-740 [32] Guyette RP, Muzika R-M, Dey DC. Dynamics of an anthropogenic fire regime. Ecosystems. 2002;5(5):472-486 [33] Hurteau MD et al. Climate change, fire management, and ecological services in the southwestern US. Forest Ecology and Management. 2014;327:280-289 [34] Mitchell JW. Power lines and catastrophic wildland fire in southern California. In: Proceedings of the 11th International Conference on Fire and Materials. Citeseer; 2009 [35] Lydersen JM, North MP, Collins BM. Severity of an uncharacteristically large wildfire, the Rim Fire, in forests with relatively restored frequent fire regimes. Forest Ecology and Management. 2014;328:326-334 [36] Prestemon JP, Butry DT. Wildland arson: A research assessment. In: Pye JM, Rauscher HM, Sands Y, Lee DC, Beatty JS, editors. Advances in Threat 40 Fire Safety and Management Awareness [1] Randerson JT et al. Global burned area and biomass burning emissions from small fires. Journal of Geophysical Research – Biogeosciences. 2012;117:G04012. DOI: [9] Harrison SP, Marlon JR, Bartlein PJ. Fire in the earth system. In: Changing Climates. Earth Systems and Society. Netherlands: Springer; 2010. pp. 21-48 [10] Marlon JR et al. Long-term perspective on wildfires in the Western USA. In: Proceedings of the National Academy of Science; 2012 [11] Torn MS, Mills E, Fried J. Will climate change spark more wildfire damage. Confronting Climate Change In California. Berkeley, CA: Lawrence Berkeley National Laboratory; 1998. [12] Gill AM, Allan G. Large fires, fire effects and the fire-regime concept. International Journal of Wildland Fire. [13] Swetnam TW, Baisan CH. Historical fire regime patterns in the southwestern United States since AD 1700. In: Fire Effects in Southwestern Forests: Proceedings of the Second La Mesa Fire Symposium, Los Alamos, New Mexico; [14] Frost CC. Presettlement fire frequency regimes of the United States: A first approximation. In: Fire in Ecosystem Management: Shifting the Paradigm from Suppression to Prescription. Tall Timbers Fire Ecology Conference Proceedings; 1998 [15] Noss RF et al. Managing fireprone forests in the western United States. Frontiers in Ecology and the Environment. 2006;4(9):481-487 [16] Parks SA et al. How will climate change affect wildland fire severity in the western US? Environmental Research Letters. 2016;11(3):035002 [17] Westerling AL et al. Warming and earlier spring increase western LBNL-42592 1994 2009;17(6):688-695 [2] National Interagency Fire Center. National Report of Wildland Fires and Acres Burned by State. 2015. Available from: https://www.nifc.gov/fireInfo/ fireInfo\_statistics.html [cited 06 May [3] Radeloff VC et al. Rapid growth of the US wildland-urban interface raises wildfire risk. Proceedings of the National Academy of Sciences. [4] Staver AC et al. Thinner bark increases sensitivity of wetter Amazonian tropical forests to fire. Ecology Letters. 2020;23(1):99-106 [5] Vines R. Heat transfer through bark, and the resistance of trees to fire. Australian Journal of Botany. [6] Melo RTM. We didn't start the fire… did we? Analyzing why California cannot seem to extinguish its worsening wildfire problem. Villanova Environmental Law Journal. [7] IPCC. In: Stocker TF, Qin D, editors. Climate Change 2013: The Physical Science Basis, Working Group I Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Summary for [8] Fried JS, Torn MS, Mills E. The impact of climate change on wildfire severity: A regional forecast for northern California. Climatic Change. 10.1029/2012JG002128 References 2018;115(13):3314-3319 1968;16(3):499-514 2020;31(1):5 Policymakers. 2013 2004;64(1-2):169-191 2015] Assessment and Their Application to Forest and Rangeland Management. Gen. Tech. Rep. PNW-GTR-802. Vol. 802. Portland, OR: US Department of Agriculture, Forest Service, Pacific Northwest and Southern Research Stations; 2010. pp. 271-283 [37] Whitlock C, Shafer SL, Marlon J. The role of climate and vegetation change in shaping past and future fire regimes in the northwestern US and the implications for ecosystem management. Forest Ecology and Management. 2003;178(1-2):5-21 [38] Pausas JG, Paula S. Fuel shapes the fire–climate relationship: Evidence from Mediterranean ecosystems. Global Ecology and Biogeography. 2012;21(11):1074-1082 [39] Haynes G, Gary H. The Early Settlement of North America: The Clovis Era. Cambridge, England: Cambridge University Press; 2002 [40] Hessburg PF, Agee JK. An environmental narrative of inland Northwest United States forests, 1800- 2000. Forest Ecology and Management. 2003;178(1-2):23-59 [41] Baker WL, Ehle D. Uncertainty in surface-fire history: The case of ponderosa pine forests in the western United States. Canadian Journal of Forest Research. 2001;31(7):1205-1226 [42] Gruell GE. Fire on the early western landscape: An annotated record of wildland fires 1776-1900. Northwest Science. 1985;59(2):97-107 [43] Busenberg G. Wildfire management in the United States: The evolution of a policy failure. Review of Policy Research. 2004;21(2):145-156 [44] Johnson JF, Bengston DN, Fan DP. US policy response to the wildfire fuels management problem: An analysis of the news media debate about the Healthy Forests Initiative and the Healthy Forests Restoration Act. Journal of Environmental Policy & Planning. 2009;11(2):129-142 [45] Addington RN et al. Relationships among wildfire, prescribed fire, and drought in a fire-prone landscape in the South-Eastern United States. International Journal of Wildland Fire. 2015;24(6):778-783 [46] Ryan KC, Knapp EE, Varner JM. Prescribed fire in North American forests and woodlands: History, current practice, and challenges. Frontiers in Ecology and the Environment. 2013;11(s1):e15-e24 [47] Prestemon JP et al. Projecting wildfire area burned in the South-Eastern United States, 2011-60. International Journal of Wildland Fire. 2016;25(7):715-729 [48] Radeloff VC et al. The wildland– urban interface in the United States. Ecological Applications. 2005;15(3):799-805 [49] Theobald DM, Romme WH. Expansion of the US wildland–urban interface. Landscape and Urban Planning. 2007;83(4):340-354 [50] Liu Z et al. Climate change and wildfire risk in an expanding wildland–urban interface: A case study from the Colorado front range corridor. Landscape Ecology. 2015;30(10):1943-1957 [51] Spyratos V, Bourgeron PS, Ghil M. Development at the wildland–urban interface and the mitigation of forest-fire risk. Proceedings of the National Academy of Sciences. 2007;104(36):14272-14276 [52] Gill AM, Stephens SL. Scientific and social challenges for the management of fire-prone wildland–urban interfaces. Environmental Research Letters. 2009;4(3):034014 43 Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… [61] Syphard AD, Brennan TJ, [62] Syphard AD, Brennan TJ, 2017;21:140-147 2013;342(6154):41-42 [64] Dupéy LN, Smith JW. An [65] Pechony O, Shindell DT. Driving forces of global wildfires over the past millennium and the forthcoming century. Proceedings of the National Academy of Sciences. 2010;107(45):19167-19170 2005;215(1-3):21-36 [66] Stephens S, Moghaddas J. [67] Wonkka CL, Rogers WE, and prescribed fire. Ecological Applications. 2015;25(8):2382-2393 Experimental fuel treatment impacts on forest structure, potential fire behavior, and predicted tree mortality in a California mixed conifer forest. Forest Ecology and Management. Kreuter UP. Legal barriers to effective ecosystem management: Exploring linkages between liability, regulations, Keeley JE. The role of defensible space for residential structure protection during wildfires. International Journal of Wildland Fire. 2014;23(8):1165-1175 Keeley JE. The importance of building construction materials relative to other factors affecting structure survival during wildfire. International Journal of Disaster Risk Reduction. [63] Stephens SL et al. Managing forests and fire in changing climates. Science. integrative review of empirical research on perceptions and behaviors related to prescribed burning and wildfire in the United States. Environmental Management. 2018;61(6):1002-1018 DOI: http://dx.doi.org/10.5772/intechopen.93245 [53] Winter G, Fried JS. Homeowner [54] Winter GJ, Vogt C, Fried JS. Fuel treatments at the wildland-urban interface: Common concerns in diverse regions. Journal of Forestry. [55] Winter G, Vogt C, McCaffrey S. Residents Warming up to Fuels Research Station; 2006. pp. 19-32 [57] Schulte S, Miller KA. Wildfire risk and climate change: The influence on homeowner mitigation behavior in the wildland–urban interface. Society and Natural Resources. 2010;23(5):417-435 [58] Egan PJ, Mullin M. Climate change: US public opinion. Annual Review of Political Science. 2017;20:209-227 [59] Dixon LS. Insurance for Wildfire Risk in California. RAND; 2018. CT-499 Testimony presented before a joint meeting of the California State Assembly and California State Senate Committees on Insurance on October 30, 2018 [60] Mell WE et al. The wildland– urban interface fire problem–current approaches and research needs. International Journal of Wildland Fire. 2010;19(2):238-251 [56] McCaffrey S et al. Social science research related to wildfire management: An overview of recent findings and future research needs. International Journal of Wildland Fire. 2013;22(1):15-24 Management: Homeowners, Acceptance of Wildfire and Fuels Management in the Wildland-Urban Interface. The public and Wildland Fire Management: Social Science Findings for Managers. Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northern perspectives on fire hazard, responsibility, and management strategies at the wildland-urban interface. Society & Natural Resources. 2000;13(1):33-49 2002;100(1):15-21 Shifting Wildfire Trends and Management Implications for the Wildland Urban Interface… DOI: http://dx.doi.org/10.5772/intechopen.93245 [53] Winter G, Fried JS. Homeowner perspectives on fire hazard, responsibility, and management strategies at the wildland-urban interface. Society & Natural Resources. 2000;13(1):33-49 Fire Safety and Management Awareness Stations; 2010. pp. 271-283 [37] Whitlock C, Shafer SL, Marlon J. The role of climate and vegetation change in shaping past and future fire regimes in the northwestern US and the implications for ecosystem management. Forest Ecology and Management. 2003;178(1-2):5-21 2012;21(11):1074-1082 2003;178(1-2):23-59 Assessment and Their Application to Forest and Rangeland Management. Gen. Tech. Rep. PNW-GTR-802. Vol. 802. Portland, OR: US Department of Agriculture, Forest Service, Pacific Northwest and Southern Research Healthy Forests Initiative and the Healthy Forests Restoration Act. Journal of Environmental Policy & Planning. [45] Addington RN et al. Relationships among wildfire, prescribed fire, and drought in a fire-prone landscape in the South-Eastern United States. International Journal of Wildland Fire. [46] Ryan KC, Knapp EE, Varner JM. Prescribed fire in North American forests and woodlands: History, current practice, and challenges. Frontiers in Ecology and the Environment. [47] Prestemon JP et al. Projecting wildfire area burned in the South-Eastern United States, 2011-60. International Journal of Wildland Fire. [48] Radeloff VC et al. The wildland– urban interface in the United States. Ecological Applications. [49] Theobald DM, Romme WH. Expansion of the US wildland–urban interface. Landscape and Urban Planning. 2007;83(4):340-354 [50] Liu Z et al. Climate change and wildfire risk in an expanding wildland–urban interface: A case study from the Colorado front range corridor. Landscape Ecology. [51] Spyratos V, Bourgeron PS, Ghil M. Development at the wildland–urban interface and the mitigation of forest-fire risk. Proceedings of the National Academy of Sciences. 2007;104(36):14272-14276 [52] Gill AM, Stephens SL. Scientific and social challenges for the management of fire-prone wildland–urban interfaces. Environmental Research Letters. 2015;30(10):1943-1957 2009;4(3):034014 2009;11(2):129-142 2015;24(6):778-783 2013;11(s1):e15-e24 2016;25(7):715-729 2005;15(3):799-805 [38] Pausas JG, Paula S. Fuel shapes the fire–climate relationship: Evidence from Mediterranean ecosystems. Global Ecology and Biogeography. [39] Haynes G, Gary H. The Early Settlement of North America: The Clovis Era. Cambridge, England: Cambridge University Press; 2002 [40] Hessburg PF, Agee JK. An environmental narrative of inland Northwest United States forests, 1800- 2000. Forest Ecology and Management. [41] Baker WL, Ehle D. Uncertainty in surface-fire history: The case of ponderosa pine forests in the western United States. Canadian Journal of Forest Research. 2001;31(7):1205-1226 [42] Gruell GE. Fire on the early western landscape: An annotated record of wildland fires 1776-1900. Northwest [43] Busenberg G. Wildfire management in the United States: The evolution of a policy failure. Review of Policy Research. 2004;21(2):145-156 [44] Johnson JF, Bengston DN, Fan DP. US policy response to the wildfire fuels management problem: An analysis of the news media debate about the Science. 1985;59(2):97-107 42 [54] Winter GJ, Vogt C, Fried JS. Fuel treatments at the wildland-urban interface: Common concerns in diverse regions. Journal of Forestry. 2002;100(1):15-21 [55] Winter G, Vogt C, McCaffrey S. Residents Warming up to Fuels Management: Homeowners, Acceptance of Wildfire and Fuels Management in the Wildland-Urban Interface. The public and Wildland Fire Management: Social Science Findings for Managers. Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northern Research Station; 2006. pp. 19-32 [56] McCaffrey S et al. Social science research related to wildfire management: An overview of recent findings and future research needs. International Journal of Wildland Fire. 2013;22(1):15-24 [57] Schulte S, Miller KA. Wildfire risk and climate change: The influence on homeowner mitigation behavior in the wildland–urban interface. Society and Natural Resources. 2010;23(5):417-435 [58] Egan PJ, Mullin M. Climate change: US public opinion. Annual Review of Political Science. 2017;20:209-227 [59] Dixon LS. Insurance for Wildfire Risk in California. RAND; 2018. CT-499 Testimony presented before a joint meeting of the California State Assembly and California State Senate Committees on Insurance on October 30, 2018 [60] Mell WE et al. The wildland– urban interface fire problem–current approaches and research needs. International Journal of Wildland Fire. 2010;19(2):238-251 [61] Syphard AD, Brennan TJ, Keeley JE. The role of defensible space for residential structure protection during wildfires. International Journal of Wildland Fire. 2014;23(8):1165-1175 [62] Syphard AD, Brennan TJ, Keeley JE. The importance of building construction materials relative to other factors affecting structure survival during wildfire. International Journal of Disaster Risk Reduction. 2017;21:140-147 [63] Stephens SL et al. Managing forests and fire in changing climates. Science. 2013;342(6154):41-42 [64] Dupéy LN, Smith JW. An integrative review of empirical research on perceptions and behaviors related to prescribed burning and wildfire in the United States. Environmental Management. 2018;61(6):1002-1018 [65] Pechony O, Shindell DT. Driving forces of global wildfires over the past millennium and the forthcoming century. Proceedings of the National Academy of Sciences. 2010;107(45):19167-19170 [66] Stephens S, Moghaddas J. Experimental fuel treatment impacts on forest structure, potential fire behavior, and predicted tree mortality in a California mixed conifer forest. Forest Ecology and Management. 2005;215(1-3):21-36 [67] Wonkka CL, Rogers WE, Kreuter UP. Legal barriers to effective ecosystem management: Exploring linkages between liability, regulations, and prescribed fire. Ecological Applications. 2015;25(8):2382-2393 Section 3 Advanced Protection Mechanism: Simulations 45 Section 3	doab	2025-04-07T03:56:59.069433	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 6 }
006df1b2-f518-4d6b-8d96-484202f13086.7	Advanced Protection Mechanism: Simulations Chapter 4 Abstract TUS were presented. 1. Introduction products. 47 physical picture of the processes. and Kamen Grozdanov Mathematical Modeling and Ivan Antonov, Rositsa Velichkova, Svetlin Antonov The mathematical models of fire distribution in a confined space–in Keywords: fire simulation, FDS, garages, buildings, numerical simulation enough and difficult for mathematical interpretation. This is due to its through Fluent and FDS using the PyroSim GUI are presented [1, 2]. Mathematical modeling and numerical simulations of fires are an essential decisive part of the solution of important problems related to fire safety, analysis of the development of fires in the investigation of their consequences. The methods that are used must have the necessary accuracy and reliability, as close as possible to the The actual fire, as it is known, is an uncontrollable combustion process, complex This chapter gives two different approaches in dealing with their complexity and implementation of solving the problem. On the other hand, an integrated, relatively simplified technical solution of a new system for preventing the spread of fires in underground garages is given, which is described in details in the chapter. The second part deals with the basic mathematical apparatus used in CFD-Fluent and FDS software. The results of two fire simulations made by the authors nonstationarity and three-dimensionality, which complicate the modeling of the heat and mass transfer processes observed in them. In the case of fires indoors of underground garages, buildings, and rooms, the development of the fire is accompanied by a change in the chronicle composition and parameters of the combustion underground garages and in buildings—are described. Integral and computational fluid mechanics methods are used. The chapter presents the results of a fire simulation using the software Fluent. It uses Reynolds-type turbulence models of the Fire Dynamic Simulation or PyroSim graphical interface with a solution model describing a turbulence. For both cases, the pictures of the spread of fire and smoke over time in an atrium of an administrative building and a five-story building of the Fires in Confined Spaces Simulation of Development of the #### Chapter 4 ## Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces Ivan Antonov, Rositsa Velichkova, Svetlin Antonov and Kamen Grozdanov #### Abstract The mathematical models of fire distribution in a confined space–in underground garages and in buildings—are described. Integral and computational fluid mechanics methods are used. The chapter presents the results of a fire simulation using the software Fluent. It uses Reynolds-type turbulence models of the Fire Dynamic Simulation or PyroSim graphical interface with a solution model describing a turbulence. For both cases, the pictures of the spread of fire and smoke over time in an atrium of an administrative building and a five-story building of the TUS were presented. Keywords: fire simulation, FDS, garages, buildings, numerical simulation #### 1. Introduction Mathematical modeling and numerical simulations of fires are an essential decisive part of the solution of important problems related to fire safety, analysis of the development of fires in the investigation of their consequences. The methods that are used must have the necessary accuracy and reliability, as close as possible to the physical picture of the processes. The actual fire, as it is known, is an uncontrollable combustion process, complex enough and difficult for mathematical interpretation. This is due to its nonstationarity and three-dimensionality, which complicate the modeling of the heat and mass transfer processes observed in them. In the case of fires indoors of underground garages, buildings, and rooms, the development of the fire is accompanied by a change in the chronicle composition and parameters of the combustion products. This chapter gives two different approaches in dealing with their complexity and implementation of solving the problem. On the other hand, an integrated, relatively simplified technical solution of a new system for preventing the spread of fires in underground garages is given, which is described in details in the chapter. The second part deals with the basic mathematical apparatus used in CFD-Fluent and FDS software. The results of two fire simulations made by the authors through Fluent and FDS using the PyroSim GUI are presented [1, 2]. #### 2. Fire extinguishing system in large underground garages: integral methods for investigation In the present part, a simple method (from a technological point of view) is offered for solution of the complex problem. It is suggested to isolate the parked in the garage cars in pairs by which will be operating a thick curtain of water at arisen burning. The necessary insulation for solid noncombustible barriers are replaced at this way [3–6]. #### 2.1 Operating principal Referring to Figure 1, cars are placed to ensure the possibility between the pairs to have enough distance for the implementation of water curtains. In case of burning over the car is formed upward convective flow, because of differences of the density of the products of combustion and the environment. This stream is proportional to the lift force: $$dF\_A = -\left[\int\_f (\rho - \rho\_{ob})gdf\right]d\mathfrak{x},\tag{1}$$ where f is the area of fire ignition and dx is elementary stretch in the vertical direction. The power of convective updraft is determined by the number of Archimedes: $$Ar = \left(\frac{\rho\_{ok}}{\rho} - 1\right) \frac{gd\_h}{u\_0^2},\tag{2}$$ where dh is the hydraulic diameter of the outbreak of fire and u<sup>0</sup> is the initial value of the velocity of the upward flow. The velocity is determined according to [6]: $$ u\_0^2 = \mathbf{1}, \Re Q^{\dagger \ddagger},\tag{3}$$ The convective flow that is formed is shown in Figure 2. The conditional flow (Figure 3). The ambient air enters the fire zone from all directions, which heats and reverses the direction vertically. The second zone is a free convective flow that continues until it reaches the ceiling of the room where the flow changes character (zone III). In this zone, the jet is transformed into a radially semi-enclosed stream The system includes fire sprinklers—quick response and standard sprinklers. Convective flow is reaching the garage ceiling under the influence of its temperature and a quick response sprinkler is switched on and the burning car is flushed with a water spray. Thus begins the process of extinguishing a fire in the initial stages. Further, propagating as a radial semi-closed jet, it reaches the "standard" This stage is defined as the isolation of burning cars from the surrounding area For the purpose of solving the task is used an integral method according to can be divided into the following areas: Convective flow is formed in zone I Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 reaction sprinklers that are included on the water curtain [7, 8]. 2.2 Mathematical model of convective non-isothermal jet [9, 10]. The used equations are as follows [11–13]: and spreads over the garage ceiling (zone IV). and no other pairs are affected. Figure 2. Figure 3. 49 Sketch of the convective flow. Distribution of the fire. where Q, kW is the power of the fire. Figure 1. Distribution of cars in the garages. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 Figure 3. Sketch of the convective flow. Figure 2. 2. Fire extinguishing system in large underground garages: integral In the present part, a simple method (from a technological point of view) is offered for solution of the complex problem. It is suggested to isolate the parked in the garage cars in pairs by which will be operating a thick curtain of water at arisen burning. The necessary insulation for solid noncombustible barriers are replaced at Referring to Figure 1, cars are placed to ensure the possibility between the pairs to have enough distance for the implementation of water curtains. In case of burning over the car is formed upward convective flow, because of differences of the density of the products of combustion and the environment. This stream is ð 2 6 4 ρ � ρok ð Þgdf 3 7 5 dx, (1) , (2) 5, (3) f Ar <sup>¼</sup> <sup>ρ</sup>ok u2 where Q, kW is the power of the fire. of the velocity of the upward flow. The velocity is determined according to [6]: <sup>0</sup> <sup>¼</sup> 1, 9Q<sup>1</sup> where f is the area of fire ignition and dx is elementary stretch in the vertical The power of convective updraft is determined by the number of Archimedes: <sup>ρ</sup> � <sup>1</sup> � � gdh where dh is the hydraulic diameter of the outbreak of fire and u<sup>0</sup> is the initial value = u2 0 dFA ¼ � methods for investigation Fire Safety and Management Awareness this way [3–6]. direction. Figure 1. 48 Distribution of cars in the garages. 2.1 Operating principal proportional to the lift force: The convective flow that is formed is shown in Figure 2. The conditional flow can be divided into the following areas: Convective flow is formed in zone I (Figure 3). The ambient air enters the fire zone from all directions, which heats and reverses the direction vertically. The second zone is a free convective flow that continues until it reaches the ceiling of the room where the flow changes character (zone III). In this zone, the jet is transformed into a radially semi-enclosed stream and spreads over the garage ceiling (zone IV). The system includes fire sprinklers—quick response and standard sprinklers. Convective flow is reaching the garage ceiling under the influence of its temperature and a quick response sprinkler is switched on and the burning car is flushed with a water spray. Thus begins the process of extinguishing a fire in the initial stages. Further, propagating as a radial semi-closed jet, it reaches the "standard" reaction sprinklers that are included on the water curtain [7, 8]. This stage is defined as the isolation of burning cars from the surrounding area and no other pairs are affected. #### 2.2 Mathematical model of convective non-isothermal jet For the purpose of solving the task is used an integral method according to [9, 10]. The used equations are as follows [11–13]: • for amount of movement $$\frac{d}{d\mathfrak{x}}\int\_{0}^{b} \rho u^{2}(\mathfrak{x}\mathfrak{y}^{j})d\mathfrak{y} = -\mathfrak{g}\int\_{0}^{b} (\rho - \rho\_{\text{ox}})(\mathfrak{x}\mathfrak{y})^{j}d\mathfrak{y} \tag{4}$$ • to preserve enthalpy flow $$\frac{d}{dx}\int\_{0}^{b} \rho \Delta h u y^{j} dy = 0\tag{5}$$ These values correspond to the case at <sup>x</sup> <sup>¼</sup> <sup>x</sup> extinguishing stream will flow over the burning car. be always greater than the above values. DOI: http://dx.doi.org/10.5772/intechopen.91274 Table 1. by the expression: where, case is isothermal. (Figure 4). Table 1. Table 2. 51 287 J=kgK, <sup>T</sup><sup>0</sup> <sup>¼</sup> 600 K, <sup>p</sup> <sup>¼</sup> <sup>10</sup><sup>5</sup> created from a burning car D<sup>0</sup> ¼ 0:5 m and height H ¼ 3÷4:5m, x of the garage will The initial velocity calculated by Eq. (3) is u<sup>0</sup> ¼ 8:2 m=s and the time when the convective stream will reach the ceiling at different heights of the garage is given in This means that less than 1 s sprinklers over the burning car will be activated and The expansion (increasing of thickness) of the jet in height can be determined dx <sup>¼</sup> <sup>0</sup>:<sup>22</sup> <sup>ρ</sup><sup>0</sup> <sup>þ</sup> <sup>ρ</sup>ок <sup>b</sup> <sup>¼</sup> <sup>0</sup>:<sup>22</sup> <sup>ρ</sup><sup>0</sup> <sup>þ</sup> <sup>ρ</sup>ок The density of the jet in the opening section is defined by Eq. (8): at R ¼ The density of the environment is <sup>ρ</sup>env <sup>¼</sup> <sup>11</sup>:2 kg=m<sup>3</sup> at the same pressure and temperature Tenv ¼ 293 K. At this density, the widening of the jet in the present For a different height in the garage, the parameter b is given in Table 2. The last row in Table 2 is given the extension of the isothermal jetð Þ T<sup>0</sup> ≈Tenv . Obviously a slight extension of non-isothermal convective flow comparing with the h, m 3 3.5 4 4.5 Δt 0.36 0.43 0.49 0.55 h (m) 3 3.5 4 4.5 b1 (m) 0.501 0.507 0.652 0.73 bsou (m) 0.651 0.77 7.74 1.98 h—height of the garage, m; b0—initial width of the radial jet, m; b1—width of the radial jet, m. h—height of the garage, m; Δt—time of the fire to reach the ceiling, m. Reaching the ceiling vertical, the convective stream is transformed into radial jet 2ρок At the relatively short distance to the ceiling, the high power of fire (the accepted conditions are Q ¼ 1500W and T ¼ 600K), the velocity and the temper- Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces ature of the rising convective stream do not change significantly. db <sup>D</sup><sup>0</sup> ≥ 3÷3:5. When adopted fire size , (11) <sup>2</sup>ρок (12) Pa in which for <sup>ρ</sup><sup>0</sup> is received <sup>ρ</sup> <sup>¼</sup> <sup>0</sup>:58 kg=m3. b ¼ 0:163x (13) • for export of vertical upward mass flow $$\frac{d}{d\mathfrak{x}}\limits\_{\mathfrak{l}}^{b}u(\rho-\rho\_{\alpha\mathbf{x}})(\pi\mathfrak{y})^{j}d\mathfrak{y}=\mathbf{0}\tag{6}$$ A simple solution can be made as (5) of enthalpy is replaced by a linear dependence on the widening of the jet $$b = c\mathfrak{x} \tag{7}$$ On the right-hand side of Eq. (4) is written the Archimedes buoyancy. The significance of included symbols is as follows: u is the jet velocity; y is the transverse coordinate; ρ is the current density; ρок is the density of the environment; and Δh is the enthalpy of the stream. The exponent j signifies: at j = 0 a flat stream and j = 1 an axis jet. The coordinate x is directed vertically upward. There is a correlation between density and temperature: $$ \rho = \frac{p}{RT}, \tag{8} $$ where p is the pressure of the environment, R is the gas constant, and T is the absolute temperature. Similarity to transverse distribution of the velocity and the density (temperature) are initiated [1, 2], where solving Eqs. (4) and (6) leads to the parameters of the upward convective stream: • the velocity of the upward stream $$ \mu\_m = B\_u^{\prime \prime} D\_0^{1\_\circ} \Delta T\_{ni}^{4\_\circ} \overline{\mathfrak{X}}^{4\_\circ} \tag{9} $$ • the temperature difference $$ \Delta T\_m = T\_m - T\_{env} = B\_{\Delta T}^{\prime \prime} D\_0^{1\natural} \Delta T^{\prime \natural} \overline{\mathfrak{X}}^{\natural \natural},\tag{10} $$ where D<sup>0</sup> is the initial diameter of the heat source of fire (the burning car); <sup>Δ</sup>T<sup>1</sup> <sup>¼</sup> Tfl � <sup>Т</sup>env; <sup>x</sup> <sup>¼</sup> <sup>x</sup> D<sup>0</sup> ; the constants B<sup>00</sup> <sup>u</sup> and B<sup>00</sup> <sup>Δ</sup><sup>T</sup> have values B<sup>00</sup> <sup>u</sup> <sup>¼</sup> <sup>0</sup>:<sup>222</sup> <sup>m</sup><sup>3</sup>K<sup>9</sup>=4 � �, B00 <sup>Δ</sup><sup>T</sup> <sup>¼</sup> <sup>0</sup>:<sup>71</sup> <sup>m</sup><sup>1</sup>=<sup>3</sup>K<sup>9</sup>=<sup>8</sup> � �. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 These values correspond to the case at <sup>x</sup> <sup>¼</sup> <sup>x</sup> <sup>D</sup><sup>0</sup> ≥ 3÷3:5. When adopted fire size created from a burning car D<sup>0</sup> ¼ 0:5 m and height H ¼ 3÷4:5m, x of the garage will be always greater than the above values. At the relatively short distance to the ceiling, the high power of fire (the accepted conditions are Q ¼ 1500W and T ¼ 600K), the velocity and the temperature of the rising convective stream do not change significantly. The initial velocity calculated by Eq. (3) is u<sup>0</sup> ¼ 8:2 m=s and the time when the convective stream will reach the ceiling at different heights of the garage is given in Table 1. This means that less than 1 s sprinklers over the burning car will be activated and extinguishing stream will flow over the burning car. The expansion (increasing of thickness) of the jet in height can be determined by the expression: $$\frac{db}{d\mathbf{x}} = \mathbf{0}.22 \frac{\rho\_0 + \rho\_{\rm oc}}{2\rho\_{\rm oc}},\tag{11}$$ where, • for amount of movement Fire Safety and Management Awareness • to preserve enthalpy flow dence on the widening of the jet d dx ð b • for export of vertical upward mass flow 0 <sup>ρ</sup>u<sup>2</sup> <sup>π</sup>y<sup>j</sup> � �dy ¼ �<sup>g</sup> d dx ð b d dx ð b axis jet. The coordinate x is directed vertically upward. the parameters of the upward convective stream: • the velocity of the upward stream D<sup>0</sup> • the temperature difference <sup>Δ</sup>T<sup>1</sup> <sup>¼</sup> Tfl � <sup>Т</sup>env; <sup>x</sup> <sup>¼</sup> <sup>x</sup> <sup>Δ</sup><sup>T</sup> <sup>¼</sup> <sup>0</sup>:<sup>71</sup> <sup>m</sup><sup>1</sup>=<sup>3</sup>K<sup>9</sup>=<sup>8</sup> � �. B00 50 There is a correlation between density and temperature: 0 0 ρΔhuy <sup>j</sup> <sup>u</sup> <sup>ρ</sup> � <sup>ρ</sup>ок ð Þð Þ <sup>π</sup><sup>y</sup> <sup>j</sup> A simple solution can be made as (5) of enthalpy is replaced by a linear depen- On the right-hand side of Eq. (4) is written the Archimedes buoyancy. The significance of included symbols is as follows: u is the jet velocity; y is the transverse coordinate; ρ is the current density; ρок is the density of the environment; and Δh is the enthalpy of the stream. The exponent j signifies: at j = 0 a flat stream and j = 1 an <sup>ρ</sup> <sup>¼</sup> <sup>p</sup> um ¼ B<sup>00</sup> uD 1=3 <sup>0</sup> ΔT 4=9 пл x<sup>1</sup>= ΔTm ¼ Tm � Tenv ¼ B<sup>00</sup> ; the constants B<sup>00</sup> where D<sup>0</sup> is the initial diameter of the heat source of fire (the burning car); where p is the pressure of the environment, R is the gas constant, and T is the absolute temperature. Similarity to transverse distribution of the velocity and the density (temperature) are initiated [1, 2], where solving Eqs. (4) and (6) leads to > <sup>Δ</sup>TD 1=3 <sup>0</sup> ΔT<sup>8</sup>=9x5= > > <sup>Δ</sup><sup>T</sup> have values B<sup>00</sup> <sup>u</sup> and B<sup>00</sup> ð b <sup>ρ</sup> � <sup>ρ</sup>ок ð Þð Þ <sup>π</sup><sup>y</sup> <sup>j</sup> dy (4) dy ¼ 0 (5) dy ¼ 0 (6) b ¼ cx (7) RT , (8) <sup>3</sup> (9) 3, (10) <sup>u</sup> <sup>¼</sup> <sup>0</sup>:<sup>222</sup> <sup>m</sup><sup>3</sup>K<sup>9</sup> =4 � �, 0 $$b = \left[0.22 \frac{\rho\_0 + \rho\_{\text{ox}}}{2\rho\_{\text{ox}}}\right] \tag{12}$$ The density of the jet in the opening section is defined by Eq. (8): at R ¼ 287 J=kgK, <sup>T</sup><sup>0</sup> <sup>¼</sup> 600 K, <sup>p</sup> <sup>¼</sup> <sup>10</sup><sup>5</sup> Pa in which for <sup>ρ</sup><sup>0</sup> is received <sup>ρ</sup> <sup>¼</sup> <sup>0</sup>:58 kg=m3. The density of the environment is <sup>ρ</sup>env <sup>¼</sup> <sup>11</sup>:2 kg=m<sup>3</sup> at the same pressure and temperature Tenv ¼ 293 K. At this density, the widening of the jet in the present case is $$b = 0.163x \tag{13}$$ For a different height in the garage, the parameter b is given in Table 2. The last row in Table 2 is given the extension of the isothermal jetð Þ T<sup>0</sup> ≈Tenv . Obviously a slight extension of non-isothermal convective flow comparing with the isothermal. Reaching the ceiling vertical, the convective stream is transformed into radial jet (Figure 4). Table 1. h—height of the garage, m; Δt—time of the fire to reach the ceiling, m. #### Table 2. h—height of the garage, m; b0—initial width of the radial jet, m; b1—width of the radial jet, m. Due to the weak widening of the jet and the short distance to the ceiling, the mass flow is not increased significantly because the temperature, density, and relatively low mileage ceiling have not changed. The jet has retained its temperature and density, and the velocity according to [14] may be determined: $$ u\_{\text{max}} = \mathbf{0}.88u\_0,\tag{14}$$ According to Figure 4, it is assumed D<sup>0</sup> ¼ b<sup>1</sup> that is already known and for flow b0 <sup>0</sup> <sup>¼</sup> <sup>Q</sup> πD<sup>0</sup> Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces The cross-section of the radial jet as a function of r is determined by the S ¼ 2πrb<sup>0</sup> where r is the current radius, b<sup>0</sup> is the width of the jet to the corresponding r. Since the resulting stream is parietal and has parietal boundary layer whose The width b<sup>0</sup> is calculated by Eq. (12) and for the case in Eq. (13) by replacing x 0 r;c > 0 r S ¼ 2:2πc , Eq. (19) can be recast in the form: <sup>0</sup> is calculated: : (18) , (19) S ¼ 2:2rb<sup>0</sup> (20) 2ρok <sup>2</sup> (22) , (21) <sup>0</sup> <sup>¼</sup> <sup>0</sup>:<sup>22</sup> <sup>ρ</sup><sup>o</sup> <sup>þ</sup> <sup>ρ</sup>ok rate in Eq. (16), the original width of the radial jet b<sup>0</sup> DOI: http://dx.doi.org/10.5772/intechopen.91274 The relationship b0ð Þ¼ x f xð Þ is given in Figure 6. <sup>b</sup><sup>0</sup> <sup>¼</sup> <sup>0</sup>:<sup>22</sup> <sup>ρ</sup><sup>0</sup> <sup>þ</sup> <sup>ρ</sup>ok When substituted in Eq. (19) we get the following: <sup>2</sup>ρok <sup>r</sup> <sup>¼</sup> <sup>c</sup> expression: thickness is approximately 0.1b<sup>0</sup> respectively, b<sup>0</sup> ¼ 0:163r. Change of the flow rate at different heights of the garage. Change of the initial weight at different heights of the garage. with r, then we have: Figure 5. Figure 6. 53 where for u<sup>0</sup> ¼ 8:2 m=s it is umax ¼ 7:2 m=s. It is assumed that the starting size of the radial jet is equal to that obtained in Table 2, b1, that is D<sup>0</sup> ¼ b1. Width of the radial jet b<sup>0</sup> is determined by the flow rate Q at the intersection of the reverse flow. The flow rate is amount of initial flow rates Q<sup>0</sup> and increase its height due to suction of air from the environment. The flow rate of ejecting fluid is considered proportional to the square of the relative increase in the width of the jet b1�b<sup>0</sup> b0 <sup>2</sup> and the distance x divided by the duration of the process Δt. $$Q\_{cj} \approx \left(\frac{b\_1 - b\_0}{b\_0}\right)^2 \frac{\varkappa}{\Delta t}, \text{m}^3/\text{s} \tag{15}$$ In this, total flow rate is obtained as the sum of normal and ejecting flow rate: $$Q = Q\_0 + Q\_{\neq}, \mathbf{m}^3/\mathbf{s},\tag{16}$$ where Q<sup>0</sup> ¼ u<sup>0</sup> πd<sup>2</sup> n <sup>4</sup> , when dn ¼ 0:5 m and u<sup>0</sup> ¼ 8 m=s we have: $$Q = Q\_0 + Q\_{\varepsilon j} = u\_0 \frac{\pi d\_n^2}{4} + \left(\frac{b\_1 - b\_0}{b\_0}\right)^2 \frac{\varkappa}{\Delta t} \tag{17}$$ The flow rate of the respective heights x ¼ 3; 3:5; 4; 4:5 m of the garage is shown in Figure 5 where it is defined by the relationship given in Eq. (12), respectively and in case of a leak by Eq. (13). Figure 4. Sketch of radial jet. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 According to Figure 4, it is assumed D<sup>0</sup> ¼ b<sup>1</sup> that is already known and for flow rate in Eq. (16), the original width of the radial jet b<sup>0</sup> <sup>0</sup> is calculated: $$b\_0' = \frac{Q}{\pi D\_0}.\tag{18}$$ The relationship b0ð Þ¼ x f xð Þ is given in Figure 6. The cross-section of the radial jet as a function of r is determined by the expression: $$S = 2\pi r b',\tag{19}$$ where r is the current radius, b<sup>0</sup> is the width of the jet to the corresponding r. Since the resulting stream is parietal and has parietal boundary layer whose thickness is approximately 0.1b<sup>0</sup> , Eq. (19) can be recast in the form: $$S = 2.2rb'\tag{20}$$ The width b<sup>0</sup> is calculated by Eq. (12) and for the case in Eq. (13) by replacing x with r, then we have: $$b' = \left[ 0.22 \frac{\rho\_0 + \rho\_{ok}}{2\rho\_{ok}} \right] r = c' r; c' = 0.22 \frac{\rho\_o + \rho\_{ok}}{2\rho\_{ok}},\tag{21}$$ respectively, b<sup>0</sup> ¼ 0:163r. Due to the weak widening of the jet and the short distance to the ceiling, the mass flow is not increased significantly because the temperature, density, and relatively low mileage ceiling have not changed. The jet has retained its temperature It is assumed that the starting size of the radial jet is equal to that obtained in and the distance x divided by the duration of the process Δt. In this, total flow rate is obtained as the sum of normal and ejecting flow rate: <sup>4</sup> , when dn ¼ 0:5 m and u<sup>0</sup> ¼ 8 m=s we have: πd<sup>2</sup> n 4 þ The flow rate of the respective heights x ¼ 3; 3:5; 4; 4:5 m of the garage is shown in Figure 5 where it is defined by the relationship given in Eq. (12), respectively and b<sup>1</sup> � b<sup>0</sup> b0 <sup>2</sup> <sup>Q</sup> <sup>¼</sup> <sup>Q</sup><sup>0</sup> <sup>þ</sup> <sup>Q</sup><sup>е</sup>j, m<sup>3</sup> Qej <sup>≈</sup> <sup>b</sup><sup>1</sup> � <sup>b</sup><sup>0</sup> b0 <sup>2</sup> Q ¼ Q<sup>0</sup> þ Q<sup>е</sup><sup>j</sup> ¼ u<sup>0</sup> Width of the radial jet b<sup>0</sup> is determined by the flow rate Q at the intersection of the reverse flow. The flow rate is amount of initial flow rates Q<sup>0</sup> and increase its height due to suction of air from the environment. The flow rate of ejecting fluid is considered proportional to the square of the relative increase in the width of the jet > x Δt , m<sup>3</sup> umax ¼ 0:88u0, (14) =s (15) =s, (16) <sup>Δ</sup><sup>t</sup> (17) x and density, and the velocity according to [14] may be determined: where for u<sup>0</sup> ¼ 8:2 m=s it is umax ¼ 7:2 m=s. Table 2, b1, that is D<sup>0</sup> ¼ b1. Fire Safety and Management Awareness where Q<sup>0</sup> ¼ u<sup>0</sup> in case of a leak by Eq. (13). πd<sup>2</sup> n b1�b<sup>0</sup> b0 <sup>2</sup> Figure 4. Sketch of radial jet. 52 When substituted in Eq. (19) we get the following: $$S = 2.2\pi c'r^2\tag{22}$$ Figure 5. Change of the flow rate at different heights of the garage. Figure 6. Change of the initial weight at different heights of the garage. The average velocity of the ceiling of the room depending on r is obtained by: $$ u\_m = \frac{\mathcal{Q}}{\mathcal{S}}\,,\tag{23}$$ In the vicinity of the burning car to sprinkler curtain, a distance of l≤2 m will trigger three (to five) fast sprinklers. At a longer distance, it will trigger maximum of three quick sprinklers of water curtain plus the main ones over the burning car and eventually those are lying in the range of l ¼ 4 m ceiling sprinklers so that the Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces To create a smokeless zone under a layer of smoke floating [14], air exhaust systems are designed and installed for smoke and hot gases. An exhaust ventilation system for smoke and hot gases is a scheme of safety equipment designed to perform a positive role in spin fire. The smoke is drawn in the direction of the noncarrier partition EI from a velocity of 2 m/s to 5 m/s. Standard allowed velocity of 5 m/s, but it should be taken into consideration that this velocity would affect From Abramovich [14], the density of the thermal load in the premises for the storage of combustible materials according to their purpose, is determined the heat capacity of the prevailing materials. The ventilation system to remove smoke and heat (VSRSH) has to reach its designed performance level within 60 s of receiving the command signal. Each VSRSH has to ensure receipt of sufficient fresh air that Heat transfer by convection and radiation is defined according to [3, 10]. Thermal effects are expressed by the intensity of the heat flow hnbt,W=m<sup>2</sup> to the surface of the element is determined taking into account the heat transfer by convection hnbt <sup>¼</sup> hnbt,<sup>c</sup> <sup>þ</sup> hnbt,<sup>r</sup>,W=m<sup>2</sup> hnbt,<sup>r</sup> <sup>¼</sup> <sup>Φ</sup>εmε<sup>f</sup> σ θð Þ <sup>1</sup><sup>r</sup> <sup>þ</sup> <sup>273</sup> <sup>2</sup> � ð Þ <sup>θ</sup><sup>m</sup> <sup>þ</sup> <sup>273</sup> <sup>4</sup> h i,W=m<sup>2</sup> (28) where heat transfer by convection hnbt,<sup>c</sup> is given by the relationship hnbt,<sup>c</sup> ¼ α<sup>c</sup> θ<sup>g</sup> � θ<sup>m</sup> Convection component of the intensity of the heat flow is determined by: temperature near the exposed fire element [°C]; and θ<sup>m</sup> is the surface temperature Radiating components of net heat flux per unit surface area are defined as hnbt,<sup>r</sup> ¼ The coefficient of heat transfer by convection α<sup>c</sup> is determined by the nominal curves corresponding to "temperature–time." On indirectly heated surface elements, the intensity of heat flow hnbt is determined by Eq. (16) where <sup>α</sup><sup>c</sup> <sup>¼</sup> <sup>4</sup> <sup>W</sup> hnbt,<sup>t</sup> ¼ α<sup>c</sup> θ<sup>g</sup> � θ<sup>m</sup> radiation heat transfer hnbt,<sup>r</sup> is given by the dependence: where α<sup>c</sup> is the heat transfer coefficient by convection <sup>W</sup> The coefficient of heat transfer by convection has value <sup>α</sup><sup>c</sup> <sup>¼</sup> <sup>9</sup> <sup>W</sup> <sup>Φ</sup>εm<sup>ε</sup> <sup>f</sup> σ θð Þ <sup>1</sup><sup>r</sup> <sup>þ</sup> <sup>273</sup> <sup>2</sup> � ð Þ <sup>θ</sup><sup>m</sup> <sup>þ</sup> <sup>273</sup> <sup>4</sup> h i,W=m<sup>2</sup> considering that the effects of heat transfer by radiation are included. , (26) m2 <sup>K</sup> � �; <sup>θ</sup><sup>g</sup> is the gas m2 <sup>K</sup> � �, , where: Ф is the factor of configuration, m2 <sup>K</sup> � �. � �,W=m<sup>2</sup> (27) � �,W=m<sup>2</sup> (29) number of activated sprinklers will increase [10]. DOI: http://dx.doi.org/10.5772/intechopen.91274 negatively and lead to the merging of streams of pure air. enters the room for the expense of the flue products. 2.3 Thermal impact and radiation, such as: of the element [°C]. 55 respectively: $$ \mu\_m = \frac{Q}{2.2\pi c'r^2}, \text{m/s} \tag{24} $$ Parking average velocity depending on r at the four heights is shown in Figure 7. Figure 8 shows the time to reach the appropriate distance: $$ \Delta t = \frac{r}{u\_m}, \text{s} \tag{25} $$ This means that in the first 2 s, all sprinklers at distance of 2 m away from the burning car will be triggered. For longer distances, the remote sprinklers will act at a condition if the temperature of the burning car does not decrease too quickly. For maximum calculated time of 7.7 s could not be expected too much decrease of the temperature, which leads to the conclusion that the ceiling temperature will be much greater than the starting temperature of "fast" sprinklers so that at tp ¼ 57°C or T = 330°K will always remain less than the temperature of the wall jet which initial temperature is 600°K. With the removal from the water curtain, it is possible to turn on the other ceiling sprinklers that are in the range. Figure 7. Average velocity at different heights of garage. Figure 8. Time to reach the sprinklers at different heights of garage. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 In the vicinity of the burning car to sprinkler curtain, a distance of l≤2 m will trigger three (to five) fast sprinklers. At a longer distance, it will trigger maximum of three quick sprinklers of water curtain plus the main ones over the burning car and eventually those are lying in the range of l ¼ 4 m ceiling sprinklers so that the number of activated sprinklers will increase [10]. To create a smokeless zone under a layer of smoke floating [14], air exhaust systems are designed and installed for smoke and hot gases. An exhaust ventilation system for smoke and hot gases is a scheme of safety equipment designed to perform a positive role in spin fire. The smoke is drawn in the direction of the noncarrier partition EI from a velocity of 2 m/s to 5 m/s. Standard allowed velocity of 5 m/s, but it should be taken into consideration that this velocity would affect negatively and lead to the merging of streams of pure air. From Abramovich [14], the density of the thermal load in the premises for the storage of combustible materials according to their purpose, is determined the heat capacity of the prevailing materials. The ventilation system to remove smoke and heat (VSRSH) has to reach its designed performance level within 60 s of receiving the command signal. Each VSRSH has to ensure receipt of sufficient fresh air that enters the room for the expense of the flue products. #### 2.3 Thermal impact The average velocity of the ceiling of the room depending on r is obtained by: um <sup>¼</sup> <sup>Q</sup> Parking average velocity depending on r at the four heights is shown in Figure 7. <sup>Δ</sup><sup>t</sup> <sup>¼</sup> <sup>r</sup> um This means that in the first 2 s, all sprinklers at distance of 2 m away from the burning car will be triggered. For longer distances, the remote sprinklers will act at a condition if the temperature of the burning car does not decrease too quickly. For maximum calculated time of 7.7 s could not be expected too much decrease of the temperature, which leads to the conclusion that the ceiling temperature will be much greater than the starting temperature of "fast" sprinklers so that at tp ¼ 57°C or T = 330°K will always remain less than the temperature of the wall jet which With the removal from the water curtain, it is possible to turn on the other um <sup>¼</sup> <sup>Q</sup> 2:2πc<sup>0</sup> Figure 8 shows the time to reach the appropriate distance: respectively: Fire Safety and Management Awareness initial temperature is 600°K. Figure 7. Figure 8. 54 ceiling sprinklers that are in the range. Average velocity at different heights of garage. Time to reach the sprinklers at different heights of garage. <sup>S</sup> , (23) <sup>r</sup><sup>2</sup> , m=<sup>s</sup> (24) , s (25) Heat transfer by convection and radiation is defined according to [3, 10]. Thermal effects are expressed by the intensity of the heat flow hnbt,W=m<sup>2</sup> to the surface of the element is determined taking into account the heat transfer by convection and radiation, such as: $$h\_{nbt} = h\_{nbt, \varepsilon} + h\_{nbt, r} \text{ W/m}^2,\tag{26}$$ where heat transfer by convection hnbt,<sup>c</sup> is given by the relationship $$h\_{\rm nbt,c} = a\_c (\theta\_\text{g} - \theta\_\text{m}) \text{, W/m}^2 \tag{27}$$ radiation heat transfer hnbt,<sup>r</sup> is given by the dependence: $$h\_{nbt,r} = \Phi \varepsilon\_m \varepsilon\_f \sigma \left[ \left( \theta\_1 r + 273 \right)^2 - \left( \theta\_m + 273 \right)^4 \right], \text{W/m}^2 \tag{28}$$ Convection component of the intensity of the heat flow is determined by: $$h\_{nbt,t} = a\_c (\theta\_\text{g} - \theta\_m), \text{W/m}^2 \tag{29}$$ where α<sup>c</sup> is the heat transfer coefficient by convection <sup>W</sup> m2 <sup>K</sup> � �; <sup>θ</sup><sup>g</sup> is the gas temperature near the exposed fire element [°C]; and θ<sup>m</sup> is the surface temperature of the element [°C]. The coefficient of heat transfer by convection α<sup>c</sup> is determined by the nominal curves corresponding to "temperature–time." On indirectly heated surface elements, the intensity of heat flow hnbt is determined by Eq. (16) where <sup>α</sup><sup>c</sup> <sup>¼</sup> <sup>4</sup> <sup>W</sup> m2 <sup>K</sup> � �. The coefficient of heat transfer by convection has value <sup>α</sup><sup>c</sup> <sup>¼</sup> <sup>9</sup> <sup>W</sup> m2 <sup>K</sup> � �, considering that the effects of heat transfer by radiation are included. Radiating components of net heat flux per unit surface area are defined as hnbt,<sup>r</sup> ¼ <sup>Φ</sup>εm<sup>ε</sup> <sup>f</sup> σ θð Þ <sup>1</sup><sup>r</sup> <sup>þ</sup> <sup>273</sup> <sup>2</sup> � ð Þ <sup>θ</sup><sup>m</sup> <sup>þ</sup> <sup>273</sup> <sup>4</sup> h i,W=m<sup>2</sup> , where: Ф is the factor of configuration, <sup>ε</sup><sup>m</sup> is the emitting surface element, <sup>ε</sup> <sup>f</sup> the transmission of fire, <sup>σ</sup> <sup>¼</sup> <sup>5</sup>:<sup>67</sup> � <sup>10</sup><sup>8</sup> WK4 m2 h i is the constant of Stefan-Boltzmann, θ<sup>r</sup> is the effective temperature of the radiation environment [°C], and θ<sup>m</sup> is the surface temperature of the element [°C]. Transmission of fire is equal to ε <sup>f</sup> ¼ 1. <sup>R</sup> <sup>¼</sup> <sup>δ</sup> Q ¼ 0:666μρ For the whole surface of the water curtain: DOI: http://dx.doi.org/10.5772/intechopen.91274 4–6 mH2O. space explosions. ρ ∂u ∂t þ ρu ∂u ∂x þ ρv ∂u ∂y þ ρw ρ ∂v ∂t þ ρu ∂v ∂x þ ρv ∂v ∂y þ ρw ∂v <sup>∂</sup><sup>z</sup> ¼ � <sup>∂</sup><sup>p</sup> ∂y þ μ 57 assumed: divV! <sup>¼</sup> 0, <sup>∂</sup><sup>u</sup> Equations for movements [9]. Continuity equation: for the optical density of the gas mixture. <sup>∂</sup><sup>x</sup> <sup>þ</sup> <sup>∂</sup><sup>v</sup> <sup>∂</sup><sup>y</sup> <sup>þ</sup> <sup>∂</sup><sup>w</sup> > ∂ρ ∂t þ <sup>∂</sup>ð Þ <sup>ρ</sup><sup>u</sup> ∂x þ <sup>∂</sup>ð Þ <sup>ρ</sup><sup>v</sup> ∂y þ ∂u <sup>∂</sup><sup>z</sup> ¼ � <sup>∂</sup><sup>p</sup> ∂x þ μ þ ∂ ∂y μT ∂u ∂y þ ∂v ∂x þ ∂ ∂x μT ∂u ∂y þ ∂v ∂x <sup>c</sup> <sup>¼</sup> <sup>0</sup>:<sup>51</sup> Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces Flow rate of the water curtain for 1 m<sup>2</sup> of lateral surface is defined by: rR H ffiffiffiffiffiffiffi <sup>2</sup>gh <sup>p</sup> <sup>¼</sup> <sup>0</sup>:<sup>467</sup> <sup>l</sup> Water curtains are constructed so that the entire hole is irrigated with finely dispersed water. For this purpose, sprinklers are placed over the hole and next to it. When they are placed at the top of the hole, it is possible for unprotected areas to remain through which it is possible for a penetration of hot gases to occur. Sprinkler heads that are used to spray jets are spaced 0.5 m in protecting small holes and 1.25–1.5 m in protecting large holes. For sprinkler heads which are situated at a distance greater than 3 m, it is required head pressure of the water 3. Numerical simulations: mathematical model of flow in a confined mechanics of fluids. These are the continuity equations, the Navier-Stokes equations in modification according to the Businex hypothesis (μeff = μ + μt), the energy equation (1st law of Thermodynamics), the Clapeyron equation for the gas mixture. Fire currents run at low speeds in the absence of detonation and <sup>∂</sup><sup>z</sup> � 0. The mathematical model is based on the equations used in the computational In the case of a fire without detonation, combustion, and explosions, it can be To these are added the equations for smoke propagation (smoke content) and ∂2 u ∂x<sup>2</sup> þ ∂2 v ∂x<sup>2</sup> þ � � � � � � � � <sup>∂</sup>ð Þ <sup>ρ</sup><sup>w</sup> ∂2 u ∂y<sup>2</sup> þ ∂2 v ∂y<sup>2</sup> þ � � � � ∂2 u ∂z<sup>2</sup> þ ∂ ∂z μT ∂u ∂z þ ∂w ∂x � � � � (36) ∂2 v ∂z<sup>2</sup> þ ∂ ∂z μT ∂v ∂z þ ∂w ∂x � � � � (37) <sup>∂</sup><sup>z</sup> <sup>¼</sup> <sup>J</sup> (35) <sup>þ</sup> <sup>2</sup> <sup>∂</sup> ∂x μT ∂u ∂x � � <sup>þ</sup> <sup>2</sup> <sup>∂</sup> ∂y μT ∂v ∂y � � <sup>2</sup>:<sup>8</sup> <sup>¼</sup> <sup>0</sup>:182 m (32) m2 (33) s <sup>Q</sup><sup>H</sup> <sup>¼</sup> <sup>11</sup>:2l=<sup>s</sup> (34) #### 2.4 Determination of the intensity of water curtain Because of the difficulties associated with the construction of fire walls, experiments are conducted so that these areas to be reduced to such proportions that the primarily split up do not disturb of the process. In many cases, such as in buildings of first degree of fire resistance, as already noted, firewalls did not provide the detriment of fire safety. In connection with this arises a need of using such fire barriers that could effectively limit the spread of fire and at the same time would give some freedom for internal layout of buildings with different functions, which is the case of the water curtain [15]. When calculating water curtains, the assumption must simultaneously satisfy the following conditions: Structural parts of the building to withstand the effects of fire on one side and the passage of flames or hot gases to be prevented by the transfer of heat to the unexposed side. The ability of the structural parts of the building to withstand the effects of fire on one side and prevent the transfer of heat from the exposed to the unexposed side. The transfer is limited so that it does not ignite either the unexposed surface, or any other material in the immediate vicinity. The structural element is designed to serve as a barrier against the heat and to ensure the protection of people who are close to it. The effectiveness of water curtains is assessed according to the amount of absorbed heat. It is known that the dependence of the growth temperature of the source of radiation of maximum energy moves to the side of the shorter waves. This follows from the law of Vin: $$ \lambda\_{\text{max},T}T = 0.29 = \text{const} \tag{30} $$ where λ is the wavelength in m,T is the temperature at the surface of water curtain, °K. Good enough inter-phase and heat-absorbing surfaces have water drops of size <sup>200</sup> � <sup>10</sup>�<sup>6</sup> . It is considered that in the best case, sprinklers spray water of size less then 1000 μm. #### 2.5 Required flow rate for air curtain The current has the following characteristics: density of the radiation heat flux is 1500 W/m2 ; density of the irradiation protected material is 900 W/m<sup>2</sup> ; height of the hole–4 m; length of the hole–6 m; pressure of water in sprinkler–0.6 MPa (6 atm) and the radius of the water drops–0.0006 m (600 μm). Opacity density of the curtain: $$\delta = \frac{2.303 \log q\_{\text{max}}}{q\_{\text{wp}}} = 0.51 \tag{31}$$ Thickness of the curtain: Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 $$R = \frac{\delta}{c} = \frac{0.51}{2.8} = 0.182 \text{ m} \tag{32}$$ Flow rate of the water curtain for 1 m<sup>2</sup> of lateral surface is defined by: $$Q = 0.666 \mu \rho \frac{rR}{H} \sqrt{2gh} = 0.467 \frac{l}{s} \text{ m}^2 \tag{33}$$ For the whole surface of the water curtain: <sup>ε</sup><sup>m</sup> is the emitting surface element, <sup>ε</sup> <sup>f</sup> the transmission of fire, <sup>σ</sup> <sup>¼</sup> <sup>5</sup>:<sup>67</sup> � <sup>10</sup><sup>8</sup> WK4 environment [°C], and θ<sup>m</sup> is the surface temperature of the element [°C]. Transmission of fire is equal to ε <sup>f</sup> ¼ 1. Fire Safety and Management Awareness is the case of the water curtain [15]. tion of people who are close to it. 2.5 Required flow rate for air curtain Opacity density of the curtain: Thickness of the curtain: and the radius of the water drops–0.0006 m (600 μm). absorbed heat. curtain, °K. <sup>200</sup> � <sup>10</sup>�<sup>6</sup> 1500 W/m2 56 then 1000 μm. from the law of Vin: the following conditions: 2.4 Determination of the intensity of water curtain is the constant of Stefan-Boltzmann, θ<sup>r</sup> is the effective temperature of the radiation Because of the difficulties associated with the construction of fire walls, experiments are conducted so that these areas to be reduced to such proportions that the primarily split up do not disturb of the process. In many cases, such as in buildings of first degree of fire resistance, as already noted, firewalls did not provide the detriment of fire safety. In connection with this arises a need of using such fire barriers that could effectively limit the spread of fire and at the same time would give some freedom for internal layout of buildings with different functions, which When calculating water curtains, the assumption must simultaneously satisfy Structural parts of the building to withstand the effects of fire on one side and the passage of flames or hot gases to be prevented by the transfer of heat to the unexposed side. The ability of the structural parts of the building to withstand the effects of fire on one side and prevent the transfer of heat from the exposed to the unexposed surface, or any other material in the immediate vicinity. The structural element is designed to serve as a barrier against the heat and to ensure the protec- The effectiveness of water curtains is assessed according to the amount of It is known that the dependence of the growth temperature of the source of radiation of maximum energy moves to the side of the shorter waves. This follows where λ is the wavelength in m,T is the temperature at the surface of water Good enough inter-phase and heat-absorbing surfaces have water drops of size The current has the following characteristics: density of the radiation heat flux is ; density of the irradiation protected material is 900 W/m<sup>2</sup> hole–4 m; length of the hole–6 m; pressure of water in sprinkler–0.6 MPa (6 atm) <sup>δ</sup> <sup>¼</sup> <sup>2</sup>:303 log <sup>q</sup>изл qкр . It is considered that in the best case, sprinklers spray water of size less λ max ,TT ¼ 0:29 ¼ const (30) ; height of the ¼ 0:51 (31) unexposed side. The transfer is limited so that it does not ignite either the m2 h i $$\mathbf{Q}^{H} = \mathbf{11.2l/s} \tag{34}$$ Water curtains are constructed so that the entire hole is irrigated with finely dispersed water. For this purpose, sprinklers are placed over the hole and next to it. When they are placed at the top of the hole, it is possible for unprotected areas to remain through which it is possible for a penetration of hot gases to occur. Sprinkler heads that are used to spray jets are spaced 0.5 m in protecting small holes and 1.25–1.5 m in protecting large holes. For sprinkler heads which are situated at a distance greater than 3 m, it is required head pressure of the water 4–6 mH2O. #### 3. Numerical simulations: mathematical model of flow in a confined space The mathematical model is based on the equations used in the computational mechanics of fluids. These are the continuity equations, the Navier-Stokes equations in modification according to the Businex hypothesis (μeff = μ + μt), the energy equation (1st law of Thermodynamics), the Clapeyron equation for the gas mixture. Fire currents run at low speeds in the absence of detonation and explosions. In the case of a fire without detonation, combustion, and explosions, it can be assumed: divV! <sup>¼</sup> 0, <sup>∂</sup><sup>u</sup> <sup>∂</sup><sup>x</sup> <sup>þ</sup> <sup>∂</sup><sup>v</sup> <sup>∂</sup><sup>y</sup> <sup>þ</sup> <sup>∂</sup><sup>w</sup> <sup>∂</sup><sup>z</sup> � 0. To these are added the equations for smoke propagation (smoke content) and for the optical density of the gas mixture. Continuity equation: $$\frac{\partial \rho}{\partial t} + \frac{\partial(\rho u)}{\partial \mathbf{x}} + \frac{\partial(\rho v)}{\partial y} + \frac{\partial(\rho w)}{\partial \mathbf{z}} = f \tag{35}$$ Equations for movements [9]. $$\begin{split} \rho \frac{\partial u}{\partial t} + \rho u \frac{\partial u}{\partial x} + \rho v \frac{\partial u}{\partial y} + \rho w \frac{\partial u}{\partial z} &= -\frac{\partial p}{\partial x} + \mu \left( \frac{\partial^2 u}{\partial x^2} + \frac{\partial^2 u}{\partial y^2} + \frac{\partial^2 u}{\partial z^2} \right) + 2 \frac{\partial}{\partial x} \left[ \mu\_T \frac{\partial u}{\partial x} \right] \\ &+ \frac{\partial}{\partial y} \left[ \mu\_T \left( \frac{\partial u}{\partial y} + \frac{\partial v}{\partial x} \right) \right] + \frac{\partial}{\partial x} \left[ \mu\_T \left( \frac{\partial u}{\partial z} + \frac{\partial w}{\partial x} \right) \right] \end{split} \tag{36}$$ $$\begin{split} \rho \frac{\partial v}{\partial t} + \rho u \frac{\partial v}{\partial x} + \rho v \frac{\partial v}{\partial y} + \rho w \frac{\partial v}{\partial z} &= -\frac{\partial p}{\partial y} + \mu \left( \frac{\partial^2 v}{\partial x^2} + \frac{\partial^2 v}{\partial y^2} + \frac{\partial^2 v}{\partial z^2} \right) + 2 \frac{\partial}{\partial y} \left[ \mu\_T \frac{\partial v}{\partial y} \right] \\ &+ \frac{\partial}{\partial x} \left[ \mu\_T \left( \frac{\partial u}{\partial y} + \frac{\partial v}{\partial x} \right) \right] + \frac{\partial}{\partial x} \left[ \mu\_T \left( \frac{\partial v}{\partial x} + \frac{\partial w}{\partial x} \right) \right] \end{split} \tag{37}$$ $$\begin{aligned} \rho \frac{\partial w}{\partial t} + \rho u \frac{\partial w}{\partial x} + \rho v \frac{\partial w}{\partial y} + \rho w \frac{\partial w}{\partial z} &= -\frac{\partial p}{\partial z} + \mu \left( \frac{\partial^2 w}{\partial x^2} + \frac{\partial^2 w}{\partial y^2} + \frac{\partial^2 w}{\partial z^2} \right) + 2 \frac{\partial}{\partial x} \left[ \mu\_T \frac{\partial w}{\partial x} \right] \\ &+ \frac{\partial}{\partial x} \left[ \mu\_T \left( \frac{\partial u}{\partial x} + \frac{\partial w}{\partial x} \right) \right] + \frac{\partial}{\partial y} \left[ \mu\_T \left( \frac{\partial v}{\partial x} + \frac{\partial w}{\partial y} \right) \right] \end{aligned} \tag{38}$$ Equations for heat exchange (1st law of Thermodynamics) $$\begin{aligned} \rho C\_p \left( \frac{\partial T}{\partial t} + u \frac{\partial T}{\partial \mathbf{x}} + v \frac{\partial T}{\partial \mathbf{y}} + w \frac{\partial T}{\partial \mathbf{z}} \right) &= \frac{\partial}{\partial \mathbf{x}} \left[ \left( \lambda + \lambda\_t + \lambda\_f \right) + \frac{\partial T}{\partial \mathbf{x}} \right] + \frac{\partial}{\partial \mathbf{y}} \left[ \left( \lambda + \lambda\_t + \lambda\_f \right) + \frac{\partial T}{\partial \mathbf{y}} \right] \\ &+ \frac{\partial}{\partial \mathbf{z}} \left[ \left( \lambda + \lambda\_t + \lambda\_f \right) + \frac{\partial T}{\partial \mathbf{z}} \right] + q + \varepsilon \end{aligned} \tag{39}$$ where cp is the specific heat content at constant pressure; λ is the coefficient of thermal conductivity; λ<sup>i</sup> is the coefficient of turbulent thermal conductivity; λ<sup>p</sup> is the coefficient of radiation thermal conductivity; and qv is the intensity of internal heat sources. Here, qv, can be represented by qv ¼ qvc þ qvr þ qvb, where qvk is the intensity of internal convective heat sources; qvb is the intensity of internal combustion sources; and qvr is the intensity of internal sources due to radiation heat transfer. Gas condition equation is given by: $$p = \rho TR,\tag{40}$$ <sup>ρ</sup> <sup>¼</sup> <sup>X</sup><sup>n</sup> i�1 Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces <sup>R</sup> <sup>¼</sup> <sup>X</sup><sup>n</sup> i�1 cp <sup>¼</sup> <sup>X</sup><sup>n</sup> i�1 χcpi where α<sup>i</sup> is the bulk concentration of the ith component and H<sup>i</sup> is its mass They can be considered as temperature dependent or considered permanent. The values of these parameters are determined at constant pressure (p ¼ const). The characteristic equation summarizes the main partial differential equations, which are solved sequentially in software for each of the flow parameters. The type where Φ is the dependent variable—velocity components, enthalpy, concentration of the components of the gas medium, optical density of the smoke, respectively; Г is the diffusion coefficient for the corresponding Φ; and S is the source Most often, a CFD-Fluent turbulence k � ε model is applied. In this model, the coefficient of turbulent viscosity υ<sup>t</sup> is represented by the Kolmogorov-Prandtl dependence, as the ratio of kinematic turbulent energy k and the rate of k2 ∂x � �<sup>2</sup> To close the system of equations at FDS, as in all other cases in turbulent flows, it is necessary to use appropriate models of turbulence. In this case, the large eddy þ ∂v ∂y � �<sup>2</sup> þ υ<sup>t</sup> ¼ C<sup>μ</sup> <sup>∂</sup><sup>x</sup> ð Þþ <sup>ρ</sup><sup>Φ</sup> divð Þ¼ <sup>ρ</sup>w<sup>Φ</sup> divð Þþ <sup>Г</sup>grad <sup>Φ</sup> <sup>S</sup>, (46) • gas constant concentration. dissipation ε: where 59 <sup>k</sup> <sup>¼</sup> <sup>1</sup> 2 3.3 FDS turbulence modeling • specific heat capacity DOI: http://dx.doi.org/10.5772/intechopen.91274 3.1 A characteristic equation ∂ member. The values for Eq. (46) are given in [9]. 3.2 Modeling the turbulence using CFD ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi <sup>u</sup>0<sup>2</sup> <sup>þ</sup> <sup>v</sup>0<sup>2</sup> <sup>þ</sup> <sup>w</sup>0<sup>2</sup> <sup>p</sup> ; <sup>ε</sup> <sup>¼</sup> <sup>υ</sup> <sup>∂</sup><sup>u</sup> of equation is as follows: αiρ<sup>i</sup> (43) χiRi (44) , (45) <sup>ε</sup> , (47) ∂w ∂z � �<sup>2</sup> " # (48) where R is the universal gas constant. Law for the conservation of the mass of the ith gas that is a part of the mixture is $$ \rho \frac{\partial \chi\_i}{\partial t} + \rho u \frac{\partial \chi\_i}{\partial x} + \rho v \frac{\partial \chi\_i}{\partial y} + \rho w \frac{\partial \chi\_i}{\partial z} = \frac{\partial}{\partial x} \left( \rho D \frac{\partial \chi\_i}{\partial x} \right) + \frac{\partial}{\partial y} \left( \rho D \frac{\partial \chi\_i}{\partial y} \right) + \frac{\partial}{\partial z} \left( \rho D \frac{\partial \chi\_i}{\partial x} \right) + m\_i,\tag{41} $$ where D is the diffusion coefficient, representing the sum of the coefficient of gas diffusion Di and the coefficient of turbulent diffusion Dtð Þ D ¼ Di þ Dt ; χ is the mass concentration of the ith gas; mi is the intensity of internal mass sources arising from the formation (disappearance) of molecules of a gas, a consequence of the ongoing chemical reactions of combustion in fires. The law (equation) for preserving the optical density of smoke is of the form: $$\frac{\partial D\_{on}}{\partial t} + u \frac{\partial D\_{on}}{\partial \mathbf{x}} + v \frac{\partial D\_{on}}{\partial \mathbf{y}} + w \frac{\partial D\_{on}}{\partial \mathbf{z}} = q\_D,\tag{42}$$ where Don is the smoke-generating capacity of the combustible material and qD is the intensity of the internal sources of optical density of the smoke formed by the ongoing reaction of combustion in a fire [3]. The thermophysical parameters of the mixture of gases involved and the result of combustion in a fire take into account the chemical composition of this mixture. It consists of oxygen, nitrogen, and combustion products - carbon monoxide, nitrogen, sulfur, etc., involved in the process combustible ingredients. They are defined as follows: • density of the mixture Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 $$ \rho = \sum\_{i=1}^{n} a\_i \rho\_i \tag{43} $$ • gas constant ρ ∂w ∂t þ ρu ρCp ∂T ∂t þ u ∂T ∂x þ v ∂T ∂y þ w ∂T ∂z heat sources. ρ ∂χi ∂t þ ρu ∂χi ∂x þ ρv ∂χi ∂y þ ρw ∂w ∂x þ ρv ∂w ∂y þ ρw Fire Safety and Management Awareness Gas condition equation is given by: where R is the universal gas constant. ∂χi <sup>∂</sup><sup>z</sup> <sup>¼</sup> <sup>∂</sup> ∂x ongoing chemical reactions of combustion in fires. ∂Don ∂t ongoing reaction of combustion in a fire [3]. defined as follows: 58 • density of the mixture þ u ∂Don ∂x þ v ∂w <sup>∂</sup><sup>z</sup> ¼ � <sup>∂</sup><sup>p</sup> ∂z þ μ Equations for heat exchange (1st law of Thermodynamics) ¼ ∂ ∂x þ ∂ ∂z and qvr is the intensity of internal sources due to radiation heat transfer. þ ∂ ∂x μT ∂u ∂z þ ∂w ∂x ∂2 w ∂x<sup>2</sup> þ λ þ λ<sup>t</sup> þ λ<sup>f</sup> <sup>þ</sup> where cp is the specific heat content at constant pressure; λ is the coefficient of thermal conductivity; λ<sup>i</sup> is the coefficient of turbulent thermal conductivity; λ<sup>p</sup> is the coefficient of radiation thermal conductivity; and qv is the intensity of internal Here, qv, can be represented by qv ¼ qvc þ qvr þ qvb, where qvk is the intensity of internal convective heat sources; qvb is the intensity of internal combustion sources; Law for the conservation of the mass of the ith gas that is a part of the mixture is þ ∂ ∂y <sup>ρ</sup><sup>D</sup> <sup>∂</sup>χ<sup>i</sup> ∂x where D is the diffusion coefficient, representing the sum of the coefficient of gas diffusion Di and the coefficient of turbulent diffusion Dtð Þ D ¼ Di þ Dt ; χ is the mass concentration of the ith gas; mi is the intensity of internal mass sources arising from the formation (disappearance) of molecules of a gas, a consequence of the The law (equation) for preserving the optical density of smoke is of the form: ∂Don ∂y where Don is the smoke-generating capacity of the combustible material and qD is the intensity of the internal sources of optical density of the smoke formed by the The thermophysical parameters of the mixture of gases involved and the result of combustion in a fire take into account the chemical composition of this mixture. It consists of oxygen, nitrogen, and combustion products - carbon monoxide, nitrogen, sulfur, etc., involved in the process combustible ingredients. They are þ w ∂Don λ þ λ<sup>t</sup> þ λ<sup>f</sup> <sup>þ</sup> ∂2 w ∂y<sup>2</sup> þ ∂T ∂x ∂2 w ∂z<sup>2</sup> þ ∂ ∂y μT ∂v ∂z þ ∂w ∂y þ ∂ ∂y p ¼ ρTR, (40) <sup>ρ</sup><sup>D</sup> <sup>∂</sup>χ<sup>i</sup> ∂y þ ∂ ∂z <sup>∂</sup><sup>z</sup> <sup>¼</sup> qD, (42) <sup>ρ</sup><sup>D</sup> <sup>∂</sup>χ<sup>i</sup> ∂z þ mi, (41) þ q þ ε ∂T ∂z <sup>þ</sup> <sup>2</sup> <sup>∂</sup> ∂z μT ∂w ∂z λ þ λ<sup>t</sup> þ λ<sup>f</sup> <sup>þ</sup> (38) ∂T ∂y (39) $$R = \sum\_{i=1}^{n} \chi\_i R\_i \tag{44}$$ • specific heat capacity $$\mathcal{L}\_p = \sum\_{i=1}^n \chi c\_{p\_i},\tag{45}$$ where α<sup>i</sup> is the bulk concentration of the ith component and H<sup>i</sup> is its mass concentration. The values of these parameters are determined at constant pressure (p ¼ const). They can be considered as temperature dependent or considered permanent. #### 3.1 A characteristic equation The characteristic equation summarizes the main partial differential equations, which are solved sequentially in software for each of the flow parameters. The type of equation is as follows: $$\frac{\partial}{\partial \mathbf{x}}(\rho \Phi) + \operatorname{div}(\rho w \Phi) = \operatorname{div}(\Gamma \text{grad } \Phi) + \mathbb{S},\tag{46}$$ where Φ is the dependent variable—velocity components, enthalpy, concentration of the components of the gas medium, optical density of the smoke, respectively; Г is the diffusion coefficient for the corresponding Φ; and S is the source member. The values for Eq. (46) are given in [9]. #### 3.2 Modeling the turbulence using CFD Most often, a CFD-Fluent turbulence k � ε model is applied. In this model, the coefficient of turbulent viscosity υ<sup>t</sup> is represented by the Kolmogorov-Prandtl dependence, as the ratio of kinematic turbulent energy k and the rate of dissipation ε: $$ \mu\_l = \mathcal{C}\_{\mu} \frac{k^2}{\varepsilon}, \tag{47} $$ where $$k = \frac{1}{2}\sqrt{u'^2 + v'^2 + w'^2}; \qquad \varepsilon = \overline{\nu \left[ \left(\frac{\partial u}{\partial \mathbf{x}}\right)^2 + \left(\frac{\partial v}{\partial \mathbf{y}}\right)^2 + \left(\frac{\partial w}{\partial \mathbf{z}}\right)^2 \right]} \tag{48}$$ #### 3.3 FDS turbulence modeling To close the system of equations at FDS, as in all other cases in turbulent flows, it is necessary to use appropriate models of turbulence. In this case, the large eddy simulation model [9] known in this type of task as the LES model is recommended as the most appropriate. The model is described in detail in [9]. The model of large eddy simulation is based on the following: large-scale vortices differ markedly in the course of transition from one current to another, with the small-scale structure changing slightly. The field of large-scale structures needs to be defined. Continuity of flow parameters is assumed using Leonard's so-called filtering function. For each flow parameter, a = a + a0 . Dissipative combustion processes such as viscous thermal conductivity, diffusion, and impurity transfer are modeled. What is special about the model is that the scale of the vortex structures is smaller than the size of the data network. The parameters μ, λ, and D in the equations describing the process are replaced by expressions modeling their effect. The strain rate tensor is used to determine μ. Thermal conductivity and impurity diffusion are determined by: $$\begin{aligned} \lambda\_t &= \frac{\mu\_t \mathbf{c}\_p}{pr\_t} \\ (\rho D)\_t &= \frac{\mu\_t}{\mathbf{S}\_{0t}} \\ \mu\_t &= \rho v\_t \end{aligned} \tag{49}$$ In the case of laminar heat transfer and diffusion, respectively: $$\begin{aligned} \lambda &= \frac{\mu c\_p}{pr} \\ (\rho D) &= \frac{\mu}{\mathbb{S}\_{\text{0t}}} \end{aligned} \tag{50}$$ The geometric model so drawn shows the location of the fire, that is, hazard Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces In real fires, there is a degree transition zone between lower cold smoke and The first smoke curtain signal may be calculated from the beginning of the transition zone formation. Thus, it can be assumed that forecasts using equations of After the 3D Atrium Model has been built (in the Gambit work environment), it is necessary to proceed with the "networking" procedure of the volume. Due to the large volume, it is not appropriate to use crosslinking of the elements in the same step. For this reason, a fine mesh is selected at the site of fire generation and its In the present case, triangular elements were selected for the site of fire generation and the smoke hatches for the networking of persons with step 0.3 m. For the Figure 11 shows the velocity field in the atrium in vector form. It can be seen from the figure that high velocities are observed at the site of smoke generation, this type depend on the exact application of the computer model. departure from the room, while a larger one is used far from them. both near the walls and the high part of the atrium. other walls as well as the volume of the atrium, a step of 0.5 m is chosen. generator and flue gas outlet (smoke hatches). Building with atrium subject to simulation study [20]. DOI: http://dx.doi.org/10.5772/intechopen.91274 higher hot smoke. Building fire development. Figure 10. 61 Figure 9. The process of combustion in the fire is most often implemented using the "Part of the mixture" approach. It is a scalar quantity characterizing the mass concentration of one or more components of a gas mixture at a given point in the flow. To reduce the volume of calculations, the significant memorized are two components of the mixture: mass concentration of unburned fuel and burned, respectively. Combustion products. Radiant heat transfer is calculated by the equations for the emission of sulfur-containing gases, which, in fact, implies a constraint on the problem. Large-scale models may also be used in certain cases. FDS equations use the FVM finite volume method. In addition to using the LES turbulence model, successful attempts have been made to apply the direct numerical modeling method described in [9]. FDS has been tested in a number of laboratories and institutions in the United States. The validation done shows the possibility of its application in many cases [16]. #### 4. Computer modeling and numerical simulations A detailed description of the Fluent (CFD) program interface is given in [17–19]. Development of fire in atrium space: The development of fire occurred in a certain object—the building shown in Figure 9 and Figure 10, located on Tsarigradsko shose Blvd., Sofia. The arrangement of the air exchange in the atrium space in case of fire is shown in Figure 10. Atrium air exchange was implemented, showing zones with critical parameters of radiation, smoke, and fire. It is important to note that all of the above is possible only by knowing the respective velocity or temperature field of the air in the room [5]. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 Figure 9. Building with atrium subject to simulation study [20]. Figure 10. Building fire development. simulation model [9] known in this type of task as the LES model is recommended processes such as viscous thermal conductivity, diffusion, and impurity transfer are modeled. What is special about the model is that the scale of the vortex structures is smaller than the size of the data network. The parameters μ, λ, and D in the equations describing the process are replaced by expressions modeling their effect. The strain rate tensor is used to determine μ. Thermal conductivity and impurity > <sup>λ</sup><sup>t</sup> <sup>¼</sup> <sup>μ</sup>tcp prt ð Þ <sup>ρ</sup><sup>D</sup> <sup>t</sup> <sup>¼</sup> <sup>μ</sup><sup>t</sup> μ<sup>t</sup> ¼ ρυ<sup>t</sup> <sup>λ</sup> <sup>¼</sup> <sup>μ</sup>cp pr ð Þ¼ <sup>ρ</sup><sup>D</sup> <sup>μ</sup> In the case of laminar heat transfer and diffusion, respectively: 4. Computer modeling and numerical simulations S0<sup>t</sup> S0<sup>t</sup> The process of combustion in the fire is most often implemented using the "Part of the mixture" approach. It is a scalar quantity characterizing the mass concentration of one or more components of a gas mixture at a given point in the flow. To reduce the volume of calculations, the significant memorized are two components of the mixture: mass concentration of unburned fuel and burned, respectively. Combustion products. Radiant heat transfer is calculated by the equations for the emission of sulfur-containing gases, which, in fact, implies a constraint on the problem. Large-scale models may also be used in certain cases. FDS equations use the FVM finite volume method. In addition to using the LES turbulence model, successful attempts have been made to apply the direct numerical modeling method described in [9]. FDS has been tested in a number of laboratories and institutions in the United States. The validation done shows the possibility of its application in A detailed description of the Fluent (CFD) program interface is given in [17–19]. Development of fire in atrium space: The development of fire occurred in a The arrangement of the air exchange in the atrium space in case of fire is shown in Figure 10. Atrium air exchange was implemented, showing zones with critical parameters of radiation, smoke, and fire. It is important to note that all of the above is possible only by knowing the respective velocity or temperature field of the air in certain object—the building shown in Figure 9 and Figure 10, located on The model of large eddy simulation is based on the following: large-scale vortices differ markedly in the course of transition from one current to another, with the small-scale structure changing slightly. The field of large-scale structures needs to be defined. Continuity of flow parameters is assumed using Leonard's so-called . Dissipative combustion (49) (50) as the most appropriate. The model is described in detail in [9]. filtering function. For each flow parameter, a = a + a0 diffusion are determined by: Fire Safety and Management Awareness many cases [16]. the room [5]. 60 Tsarigradsko shose Blvd., Sofia. The geometric model so drawn shows the location of the fire, that is, hazard generator and flue gas outlet (smoke hatches). In real fires, there is a degree transition zone between lower cold smoke and higher hot smoke. The first smoke curtain signal may be calculated from the beginning of the transition zone formation. Thus, it can be assumed that forecasts using equations of this type depend on the exact application of the computer model. After the 3D Atrium Model has been built (in the Gambit work environment), it is necessary to proceed with the "networking" procedure of the volume. Due to the large volume, it is not appropriate to use crosslinking of the elements in the same step. For this reason, a fine mesh is selected at the site of fire generation and its departure from the room, while a larger one is used far from them. In the present case, triangular elements were selected for the site of fire generation and the smoke hatches for the networking of persons with step 0.3 m. For the other walls as well as the volume of the atrium, a step of 0.5 m is chosen. Figure 11 shows the velocity field in the atrium in vector form. It can be seen from the figure that high velocities are observed at the site of smoke generation, both near the walls and the high part of the atrium. The temperature distribution in the volume of the atrium is shown in Figure 12. Areas with higher temperatures are clearly visible—near the source of smoke and the surrounding wall above it, and near the dome of the atrium. Figure 13 shows the distribution of smoke in the atrium at various points in time for 120 s until equilibrium between the ascending and descending currents in the atrium is reached. Figure 14 shows the change in turbulent kinetic energy in the atrium. What is striking is the fact that there is an intense transfer of substances from the outbreak of the fire along the wall of the atrium to the dome, and then it slowly subsides. When smoke reaches the floor of the room, the turbulent kinetic energy is approximately zero. Modern computer programs for numerical modeling of processes related to the simulation of air exchange in atriums can alleviate some regulatory requirements for protected premises (atriums), which can lead to significant savings for investors. If necessary, openings may be left open in the premises. With the use of fire ventilation, they will not have a negative effect on the parameters of the fire. In large areas, flue products may only be contained above the fire. The ability to make new, more practical, and economical architectural decisions is increasing. Application of the FDS environment for predicting and restoring the spread of fires and damage in the building [21, 22]. An analysis is made in the FDS environment to look at the basic features on which it is based. In analyzing the program, it should be emphasized that it is related to the numerical mechanics of the fluids and software products built on this basis. The same system of private differential equations is used, with the difference between the CFD and the FDS medium in the equations used to describe the turbulence. Fluent programs utilize turbulence models, which narrows their applicability in the study of fires in unlimited space. Large Eddy Simulation (LES) is used for FDS. This expands the applicability in the study of currents and fires in open space, as well as the effect of wind, etc. Weather conditions when solving problems. Figure 12. Figure 13. 63 The distribution of temperature in the volume of the atrium. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 The distribution of smoke in the atrium at different times. (a) 1320 s, and (b) 1440 s. Figure 11. The velocity field in atrium in vector form. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 Figure 12. The distribution of temperature in the volume of the atrium. Figure 13. The distribution of smoke in the atrium at different times. (a) 1320 s, and (b) 1440 s. The temperature distribution in the volume of the atrium is shown in Figure 12. Areas with higher temperatures are clearly visible—near the source of smoke and Figure 13 shows the distribution of smoke in the atrium at various points in time for 120 s until equilibrium between the ascending and descending currents in the Figure 14 shows the change in turbulent kinetic energy in the atrium. What is striking is the fact that there is an intense transfer of substances from the outbreak of the fire along the wall of the atrium to the dome, and then it slowly subsides. When smoke reaches the floor of the room, the turbulent kinetic energy is approximately zero. Modern computer programs for numerical modeling of processes related to the simulation of air exchange in atriums can alleviate some regulatory requirements for protected premises (atriums), which can lead to significant savings for investors. If necessary, openings may be left open in the premises. With the use of fire ventilation, they will not have a negative effect on the parameters of the fire. In large areas, flue products may only be contained above the fire. The ability to make new, more practical, and economical architectural decisions is increasing. fires and damage in the building [21, 22]. Application of the FDS environment for predicting and restoring the spread of An analysis is made in the FDS environment to look at the basic features on which it is based. In analyzing the program, it should be emphasized that it is related to the numerical mechanics of the fluids and software products built on this basis. The same system of private differential equations is used, with the difference between the CFD and the FDS medium in the equations used to describe the turbulence. Fluent programs utilize turbulence models, which narrows their applicability in the study of fires in unlimited space. Large Eddy Simulation (LES) is used for FDS. This expands the applicability in the study of currents and fires in open space, as well as the effect of wind, etc. Weather conditions when solving problems. the surrounding wall above it, and near the dome of the atrium. atrium is reached. Fire Safety and Management Awareness Figure 11. 62 The velocity field in atrium in vector form. Figure 14. Change in turbulent kinetic energy in the atrium. The program is also used to analyze the spread of hazards in the work environment, both industrial and residential sites, as well as in the environment. This program allows to restore the development of fire in past events [5, 9, 16]. #### 4.1 Closed-loop fire development modeling using the PyroSim (FDS) program This simulation product is applicable to the modeling of fire development and the determination of the evacuation and extinguishing route indoors. The software environment offers intuitive function menus (graphical user interface) and provides results for the propagation of flue gases, hydrocarbons, and other substances during a fire, as well as the temperature distribution along the cross section of the model's geometry. The program serves not only the prediction of the situation, but also the investigation of fire in the setting of the initial ignition zone, as well as training. The simulations in the program are based on the computational dynamics of fluids, and in particular, low-velocity convective currents. The capabilities of the software make it possible to investigate fires from cooking stoves to oil derivative stores (oil bases). The program is also applicable to simulation of flame-free processes, including building ventilation testing. A detailed description of how to work with the PyroSim interface is given in [17]. Development of fire in a training building: The development of a fire in study building 2 of TU-Sofia is investigated. The fire is assumed to start from the ground floor—one of the laboratories (Figure 15). Specific examples of the application of the PyroSim software product are shown in Figures 16–21 in a simulated fire in a training laboratory on the first floor of a technical building of the Technical University—Sofia. For the construction of the geometric model in Figure 16, the real barrier elements such as walls, doors, and windows, as well as the materials of which they are constructed with their respective melting/ignition temperatures, are taken into account. as fast as possible on one of the stairs, which is a kind of chimney (chimney) for this part of the building. For the same period of time, smoke spreads down the corridor on the first floor. Since there are no smoke barriers (doors) installed between the same staircase and the corridors on the floors, it will spread to all floors and will make it difficult to evacuate people in the building. Partition doors are placed on the next staircase (to the right of the model shown in Figures 18 and 19), which are intended to prevent the smoke from burning the floors in the direction from the staircase to the corridors, but in this case the flue gases will meet on both sides. The same barriers and Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces Figure 16. Figure 15. Building 2 of TU-Sofia. Figure 17. 65 Working environment for drawing the geometric model. DOI: http://dx.doi.org/10.5772/intechopen.91274 Flue gas propagation in the building within 50 s of the simulation. Instantaneous flue gas images of the building are shown in Figure 17 (for 50s), Figure 18 (for 60s), and Figure 19 (for 440 s). It is clear that the smoke is spreading Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 Figure 15. Building 2 of TU-Sofia. The program is also used to analyze the spread of hazards in the work environment, both industrial and residential sites, as well as in the environment. This program 4.1 Closed-loop fire development modeling using the PyroSim (FDS) program This simulation product is applicable to the modeling of fire development and the determination of the evacuation and extinguishing route indoors. The software environment offers intuitive function menus (graphical user interface) and provides results for the propagation of flue gases, hydrocarbons, and other substances during a fire, as well as the temperature distribution along the cross section of the model's geometry. The program serves not only the prediction of the situation, but also the investigation of fire in the setting of the initial ignition zone, as well as training. The simulations in the program are based on the computational dynamics of fluids, and in particular, low-velocity convective currents. The capabilities of the software make it possible to investigate fires from cooking stoves to oil derivative stores (oil bases). The program is also applicable to simulation of flame-free A detailed description of how to work with the PyroSim interface is given Development of fire in a training building: The development of a fire in study building 2 of TU-Sofia is investigated. The fire is assumed to start from the ground Specific examples of the application of the PyroSim software product are shown in Figures 16–21 in a simulated fire in a training laboratory on the first floor of a technical building of the Technical University—Sofia. For the construction of the geometric model in Figure 16, the real barrier elements such as walls, doors, and windows, as well as the materials of which they are constructed with their respec- Instantaneous flue gas images of the building are shown in Figure 17 (for 50s), Figure 18 (for 60s), and Figure 19 (for 440 s). It is clear that the smoke is spreading allows to restore the development of fire in past events [5, 9, 16]. processes, including building ventilation testing. tive melting/ignition temperatures, are taken into account. floor—one of the laboratories (Figure 15). in [17]. 64 Figure 14. Change in turbulent kinetic energy in the atrium. Fire Safety and Management Awareness Figure 16. Working environment for drawing the geometric model. Flue gas propagation in the building within 50 s of the simulation. as fast as possible on one of the stairs, which is a kind of chimney (chimney) for this part of the building. For the same period of time, smoke spreads down the corridor on the first floor. Since there are no smoke barriers (doors) installed between the same staircase and the corridors on the floors, it will spread to all floors and will make it difficult to evacuate people in the building. Partition doors are placed on the next staircase (to the right of the model shown in Figures 18 and 19), which are intended to prevent the smoke from burning the floors in the direction from the staircase to the corridors, but in this case the flue gases will meet on both sides. The same barriers and #### Figure 18. Flue gas propagation in the building within 60 s of the simulation. Figure 20 shows the velocity distribution along the vertical section of a building for 3800 s of the simulation. It is clear that the first and last floors of the building and the staircase adjacent to the burning room are affected at the beginning of the Figure 21 shows the temperature distribution along the vertical section of a building for the 480th second of the simulation. From here, it is reported that in the fire zone in the laboratory the temperature is above 200°C, and at the site in the hallway in front of it, where the nearby staircase is, the temperature is above 120°C. As the building climbs, the temperature drops to about 60°C until the third floor, indicating that there should be no escape route in this area without protective clothing. By linking the data from the previous figures, the instructions for the mandatory availability of respiratory protection may also be added, as this is also Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces The results of the simulation of a fire occurring in a particular building give preliminary information about the flaws in its design with respect to fire safety. If taken into account, placing barriers in the right places, as well as revising the evacuation route from the building would lead to increased security in the event of a disaster or accident and to removal all people without damage to their health. The results obtained in this chapter are first and foremost a practical application that allows solving problems related to fire prevention and analysis in a restricted area. The technical solution to limit the spread of fires is to use protective water curtains, as they isolate burning vehicles from the environment and thus prevent the transfer of fire to other vehicles in the underground garage. The solution can be The results of the two simulations of fire in specific buildings indicate the possibility of Fluent and FDS-PyroSim software in analyzing fire spread, smoke, temperature, and harmfulness in confined spaces. As shown, these simulations can be used: • in the case of designing buildings with fixed sprinklers and evacuation routes. • in judicial analysis of the consequences of the fire by initiation of its process, and then the other floors. Figure 21. 5. Conclusion 67 the main route for the distribution of flue gases. Temperature distribution for the 480th second of the simulation. DOI: http://dx.doi.org/10.5772/intechopen.91274 applied to any particular similar object. development over time. Figure 19. Flue gas propagation in the building at 440 s of the simulation. reduced visibility in this enclosure will cause additional evacuation difficulties, because people will not easily notice where the barrier on the second staircase is and are likely to collide with it glass shutter door, which is closed by a mechanical machine (mechanism), which is a prerequisite for an accident during the evacuation and may lead to an increase in the number of casualties in the building. Figure 20. Speed distribution for 3800 s of simulation. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 Figure 21. Temperature distribution for the 480th second of the simulation. Figure 20 shows the velocity distribution along the vertical section of a building for 3800 s of the simulation. It is clear that the first and last floors of the building and the staircase adjacent to the burning room are affected at the beginning of the process, and then the other floors. Figure 21 shows the temperature distribution along the vertical section of a building for the 480th second of the simulation. From here, it is reported that in the fire zone in the laboratory the temperature is above 200°C, and at the site in the hallway in front of it, where the nearby staircase is, the temperature is above 120°C. As the building climbs, the temperature drops to about 60°C until the third floor, indicating that there should be no escape route in this area without protective clothing. By linking the data from the previous figures, the instructions for the mandatory availability of respiratory protection may also be added, as this is also the main route for the distribution of flue gases. The results of the simulation of a fire occurring in a particular building give preliminary information about the flaws in its design with respect to fire safety. If taken into account, placing barriers in the right places, as well as revising the evacuation route from the building would lead to increased security in the event of a disaster or accident and to removal all people without damage to their health. #### 5. Conclusion reduced visibility in this enclosure will cause additional evacuation difficulties, because people will not easily notice where the barrier on the second staircase is and are likely to collide with it glass shutter door, which is closed by a mechanical machine (mechanism), which is a prerequisite for an accident during the evacuation and may lead to an increase in the number of casualties in the building. Flue gas propagation in the building within 60 s of the simulation. Fire Safety and Management Awareness Flue gas propagation in the building at 440 s of the simulation. Figure 18. Figure 19. Figure 20. 66 Speed distribution for 3800 s of simulation. The results obtained in this chapter are first and foremost a practical application that allows solving problems related to fire prevention and analysis in a restricted area. The technical solution to limit the spread of fires is to use protective water curtains, as they isolate burning vehicles from the environment and thus prevent the transfer of fire to other vehicles in the underground garage. The solution can be applied to any particular similar object. The results of the two simulations of fire in specific buildings indicate the possibility of Fluent and FDS-PyroSim software in analyzing fire spread, smoke, temperature, and harmfulness in confined spaces. As shown, these simulations can be used: Fire Safety and Management Awareness References 2004 [1] Puzach SV, Chumachenko AP, Kozlov YI, Bubnov VM, Rodin BC. Method of calculation with a computer program for determining the actual limits of fire resistance and modeling of actions of fire extinguishing systems. In: Mechanical Ventilation and Smoke Removal During Fires. Moscow: VDPO; DOI: http://dx.doi.org/10.5772/intechopen.91274 Conference on Thermal Equipment, Renewable Energy and Rural 151155 Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces 2016 Development. E3S Web of Conferences TE-RE-RD 2019; Targoviste; Romania; 6 June 2019 through 8 June 2019; Vol. 112. 2019. Article number 01015; Code [9] Antonov I. Applied Fluid Mechanics. Sofia: Technical University of Sofia; [10] Marshall AM, di Marzo M. Modeling aspects of sprinkler spray dynamics in fires. Process Safety and Environmental [11] Launder BE, Spalding DB. Lectures on Mathematical Models of Turbulence. [12] Leonard A. Energy cascade in largeeddy simulation of turbulent fluid flows. Protection. 2004;82(2):97-104 London: Academic Press; 1972 Advances in Geophysics. 1975;18 [13] Rodi W, Spalding DB. A twoparameter model of turbulence and its application to free jets. Warms and Stoffuberrtrag. 1970;3:585-595 [14] Abramovich GN. Theory of Turbulent Jets. Moscow; 2011. ISBN: [15] Velichkova R, Antonov I, Nikolov K, Grozdanov K, Uzunova M. Modeling of the occurrence of fire in closed cars garages. In: EFEA' 2016. DOI: 10.1109/ EFEA.2016.7748807. Available from: http://ieeexplore.ieee.org/document/ 7748807/. Electronic ISBN: 978-1- [16] Pichurov G, Stankov P, Ivanov M. Radial jet predictions based on computational fluid dynamics. In: Healthy Buildings 2006: Creating a Healthy Indoor Environment for People, Proceedings, Vol. 5. 2006. pp. 125-128. ISBN: 978-1-62276-998-8 (Part A):237-248 978-5-4365-0031- 5090-0749-3 [2] PyroSim Example Guide. 2012. Available from: https://www.thunde rheadeng.com/pyrosim/tutorials/ Grozdanov K, Uzunova M. The possibility of replacing solid walls with water curtain applicable to a large underground garage. In: EFEA'2016, Belgrade, Serbia. 2016. DOI: 10.1109/EFEA.2016.7748805. Available from: http://ieeexplore.ieee.org/ document/774880/; electronic ISBN: [4] Antonov IS. About a modification of k-ε model applicable to heat and mass transfer processes in two-phase turbulent flows. In: EMF'98 Science Conference Energy Efficiency and Environmental Protection. September 17–20, 1998, Sozopol, Proceedings. 1998. pp. 7-14 [5] CFD modeling of a large complex fire. Report 3120. Lund; 2000 [6] Drysdale D. An Introduction to Fire [7] Stoyanov V, Terziev A, Uzunova M. Numerical study of distribution of smoke and hazards in underground parking areas considering the operation of ventilation. In: 2014 3rd International Symposium on Environmental Friendly Energies and Applications (EFEA); [8] Terziev A. Study of the fire dynamics in a burning car and analysis of the possibilities for transfer of fire to a nearby vehicle. In: 8th International Dynamics. UK: Wiley; 2011 St. Ouen. 2014. pp. 1-6 69 [3] Antonov I, Velichkova R, 978-1-5090-0749-3 #### Author details Ivan Antonov, Rositsa Velichkova\, Svetlin Antonov and Kamen Grozdanov Technical Univesity of Sofia, Sofia, Bulgaria \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mathematical Modeling and Simulation of Development of the Fires in Confined Spaces DOI: http://dx.doi.org/10.5772/intechopen.91274 #### References [1] Puzach SV, Chumachenko AP, Kozlov YI, Bubnov VM, Rodin BC. Method of calculation with a computer program for determining the actual limits of fire resistance and modeling of actions of fire extinguishing systems. In: Mechanical Ventilation and Smoke Removal During Fires. Moscow: VDPO; 2004 [2] PyroSim Example Guide. 2012. Available from: https://www.thunde rheadeng.com/pyrosim/tutorials/ [3] Antonov I, Velichkova R, Grozdanov K, Uzunova M. The possibility of replacing solid walls with water curtain applicable to a large underground garage. In: EFEA'2016, Belgrade, Serbia. 2016. DOI: 10.1109/EFEA.2016.7748805. Available from: http://ieeexplore.ieee.org/ document/774880/; electronic ISBN: 978-1-5090-0749-3 [4] Antonov IS. About a modification of k-ε model applicable to heat and mass transfer processes in two-phase turbulent flows. In: EMF'98 Science Conference Energy Efficiency and Environmental Protection. September 17–20, 1998, Sozopol, Proceedings. 1998. pp. 7-14 [5] CFD modeling of a large complex fire. Report 3120. Lund; 2000 [6] Drysdale D. An Introduction to Fire Dynamics. UK: Wiley; 2011 [7] Stoyanov V, Terziev A, Uzunova M. Numerical study of distribution of smoke and hazards in underground parking areas considering the operation of ventilation. In: 2014 3rd International Symposium on Environmental Friendly Energies and Applications (EFEA); St. Ouen. 2014. pp. 1-6 [8] Terziev A. Study of the fire dynamics in a burning car and analysis of the possibilities for transfer of fire to a nearby vehicle. In: 8th International Conference on Thermal Equipment, Renewable Energy and Rural Development. E3S Web of Conferences TE-RE-RD 2019; Targoviste; Romania; 6 June 2019 through 8 June 2019; Vol. 112. 2019. Article number 01015; Code 151155 [9] Antonov I. Applied Fluid Mechanics. Sofia: Technical University of Sofia; 2016 [10] Marshall AM, di Marzo M. Modeling aspects of sprinkler spray dynamics in fires. Process Safety and Environmental Protection. 2004;82(2):97-104 [11] Launder BE, Spalding DB. Lectures on Mathematical Models of Turbulence. London: Academic Press; 1972 [12] Leonard A. Energy cascade in largeeddy simulation of turbulent fluid flows. Advances in Geophysics. 1975;18 (Part A):237-248 [13] Rodi W, Spalding DB. A twoparameter model of turbulence and its application to free jets. Warms and Stoffuberrtrag. 1970;3:585-595 [14] Abramovich GN. Theory of Turbulent Jets. Moscow; 2011. ISBN: 978-5-4365-0031- [15] Velichkova R, Antonov I, Nikolov K, Grozdanov K, Uzunova M. Modeling of the occurrence of fire in closed cars garages. In: EFEA' 2016. DOI: 10.1109/ EFEA.2016.7748807. Available from: http://ieeexplore.ieee.org/document/ 7748807/. Electronic ISBN: 978-1- 5090-0749-3 [16] Pichurov G, Stankov P, Ivanov M. Radial jet predictions based on computational fluid dynamics. In: Healthy Buildings 2006: Creating a Healthy Indoor Environment for People, Proceedings, Vol. 5. 2006. pp. 125-128. ISBN: 978-1-62276-998-8 Author details 68 Technical Univesity of Sofia, Sofia, Bulgaria Fire Safety and Management Awareness provided the original work is properly cited. \Address all correspondence to: [email protected] Ivan Antonov, Rositsa Velichkova\, Svetlin Antonov and Kamen Grozdanov © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, [17] Antonov SV, Antonov IV, Grozdanov K. Modelling and Simulation of Fire. Sofia: Technical University of Sofia; 2018 Chapter 5 Abstract underground mines. 1. Introduction modeling 71 Scenarios Methodology for Optimal Fire The purpose of this chapter is to develop a methodology that will contribute in locating optimal evacuation routes in case of fire that are based on minimal carbon monoxide (CO) exposure during the evacuation procedures. The proposed methodology is tested using simulated fire scenarios from which CO concentration over time curve is extracted from all available evacuation routes and presented in a weighted form based on the accumulating effect of CO inhalation in the form of fractional effective dose (FED). The safety limits of the FED model on which the optimization process is based are determined using a model for the prediction of carboxyhemoglobin (COHb) levels in human blood. The COHb model is associated with predicted clinical symptoms that are the basis for determining the level of incapacitation at which the mineworkers are incapable of completing their evacuation. Also in the process of improving the fire risk analysis, the proposed methodology enables the development of evacuation plans that are based on the results of modeled fire scenarios combined together with the results of the anticipated hazards generated by CO inhalation. The results presented in this chapter indicate a more precise approach in the process of planning the evacuation system inside the Keywords: underground mines, fire, safety, evacuation, optimization, simulation, Fires are one of the most serious accidents that can occur in underground mines due to the restricted ability to evacuate quickly from the confined excavations that can be filled quickly with smoke and noxious fumes [1]. The behavior of underground mine fires is difficult to predict due to their dependence on multiple factors that are closely related to the amount of flammable material, ignition location, ventilation system arrangement, time of occurrence, etc. [2]. These uncertainties associated with mine fire scenarios can have unexpected impacts on the evacuation process, firefighting, and rescue strategies and also further complicate the process of design and implementation of fire protection systems. Evacuations in Underground Mines Based on Simulated Vancho Adjiski and Zoran Despodov [18] Antonov I, Terziev A. Tutorial on Applied Fluid Mechanics. Sofia: Technical University of Sofia; 2012 [19] Fluent Inc. Chapter 10. Modeling turbulence. UK: Fluent; 2001 [20] Grozdanov K. TS. Modeling of fire in auto accident with the purpose of event identification [PhD thesis]. Sofia; 2017 [21] FDS-SMV Official Website. Ford Dynamics Simulator and Smokeview. Available from: https://pages.nist.gov/ fds-smv/ [22] Puzach SV. Mathematical Modeling of Gas Dynamics and Heat and Mass Transfer in Solving Fire Safety Problems. Moscow: Russian Academy of Medical Science; 2003 #### Chapter 5 [17] Antonov SV, Antonov IV, Fire Safety and Management Awareness Sofia; 2018 fds-smv/ 70 Grozdanov K. Modelling and Simulation of Fire. Sofia: Technical University of [18] Antonov I, Terziev A. Tutorial on Applied Fluid Mechanics. Sofia: Technical University of Sofia; 2012 [19] Fluent Inc. Chapter 10. Modeling [20] Grozdanov K. TS. Modeling of fire in auto accident with the purpose of event identification [PhD thesis]. Sofia; 2017 [21] FDS-SMV Official Website. Ford Dynamics Simulator and Smokeview. Available from: https://pages.nist.gov/ [22] Puzach SV. Mathematical Modeling of Gas Dynamics and Heat and Mass Transfer in Solving Fire Safety Problems. Moscow: Russian Academy of Medical Science; 2003 turbulence. UK: Fluent; 2001 ## Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios Vancho Adjiski and Zoran Despodov ## Abstract The purpose of this chapter is to develop a methodology that will contribute in locating optimal evacuation routes in case of fire that are based on minimal carbon monoxide (CO) exposure during the evacuation procedures. The proposed methodology is tested using simulated fire scenarios from which CO concentration over time curve is extracted from all available evacuation routes and presented in a weighted form based on the accumulating effect of CO inhalation in the form of fractional effective dose (FED). The safety limits of the FED model on which the optimization process is based are determined using a model for the prediction of carboxyhemoglobin (COHb) levels in human blood. The COHb model is associated with predicted clinical symptoms that are the basis for determining the level of incapacitation at which the mineworkers are incapable of completing their evacuation. Also in the process of improving the fire risk analysis, the proposed methodology enables the development of evacuation plans that are based on the results of modeled fire scenarios combined together with the results of the anticipated hazards generated by CO inhalation. The results presented in this chapter indicate a more precise approach in the process of planning the evacuation system inside the underground mines. Keywords: underground mines, fire, safety, evacuation, optimization, simulation, modeling ## 1. Introduction Fires are one of the most serious accidents that can occur in underground mines due to the restricted ability to evacuate quickly from the confined excavations that can be filled quickly with smoke and noxious fumes [1]. The behavior of underground mine fires is difficult to predict due to their dependence on multiple factors that are closely related to the amount of flammable material, ignition location, ventilation system arrangement, time of occurrence, etc. [2]. These uncertainties associated with mine fire scenarios can have unexpected impacts on the evacuation process, firefighting, and rescue strategies and also further complicate the process of design and implementation of fire protection systems. Developing effective evacuation plans in case of fire in underground mine is the most important and sometimes the only option for safe evacuation of all involved in the fire scenario. The wide range of possibilities in the process of improving the evacuation plans in case of fire has motivated many researchers to make new or to modify the existing methodologies or procedures for developing effective and optimal evacuation plans. • Analysis of production plans DOI: http://dx.doi.org/10.5772/intechopen.91213 the required production capacity. the necessary steps presented on Figure 1. the underground mine are also marked. Methodology for developing and locating fire scenarios in underground mines. Figure 1. 73 • Analysis of work processes and mechanization, etc. The dynamics of mining activities to increase and fulfill production capacity generates a constant shift in production sites generally associated with mechanization that is likely to trigger a fire scenario. Due to this fact as a relevant indicator that realistically reflects and constantly updates, the list of possible fire locations would be a detailed analysis of daily or monthly production plans. This step involves a thorough analysis of the daily/monthly production plans that will detect any flammable materials mostly associated with the mechanization needed to achieve Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios A case study of the "SASA"-R.N. Macedonia mine was used in order to conduct The steps shown in Figure 1 are based on a simple analysis of the production plans that can detect all workplaces with the appropriate work cycle together with To demonstrate the presented methodology, a 3D model of the underground ventilation network of the mine "SASA"-R.N. Macedonia is prepared on which all the necessary analysis and simulations will be performed (Figure 2). On the ventilation map, the possible fire locations along with the group of mineworkers identified using the proposed methodology on Figure 1 and also the possible exits from The process of modeling fire scenarios is closely related to the degree of uncertainty when it comes to the input data, which largely depends on the size of the fire itself [11, 12]. Examples of such input parameters that affect the fire models in underground mines are fire load, fire location, burn rate of materials, heat release rate, ventilation parameters, etc. Due to the stochastic nature of the input parameters related to the fire models, the appropriate results should be treated with caution. From the large list of stochastic input parameters, the authors decided to elaborate only on the process of obtaining fire load inputs, which largely depends on the related mechanization which is often associated with fire scenarios. Ji et al. [3] developed a visual model to simulate the evacuation process of miners to determine the evacuation time, exit flow rate, and evacuation path and show that simulation is effective technology to establish safe evacuation system. Chen et al. [4] developed 3D CFD model to reconstruct the laneway conveyor belt fire scenes under two ventilating conditions to investigate the influence of smoke movement on miner evacuation behaviors. Wang et al. [5] through example demonstrated the use of their proposed framework for human error risk analysis of coal mine emergency evacuation and also the method to evaluate the reliability of human safety barriers. Wu et al. [6] conducted emergency evacuation simulation and visualized analysis of underground mine water bursting disaster scene, to achieve the simulation of the dynamic process of individual or group behavior and to provide platform for rational evacuation under the situation of mine disaster. Adjiski et al. [7–9] completed many different manuscripts and projects in the field of simulation and modeling of fire scenarios and evacuation plans in underground mines. To the authors' best knowledge and the extensive search of literature, a lack of methodologies and systems that focus on developing evacuation plans in case of fire in underground mines is shown. Due to the large number of factors from which the effective evacuation process depends, this field of research requires continuous upgrading to address all challenges and also to provide optimal evacuation routes that sometimes represent the only option for preventing loss of human lives. This chapter is an extension and upgrade of the previously published works from the same author and hopefully will contribute to the process that will improve the methodologies and systems for optimal fire evacuations in underground mines. #### 2. Methodology for developing underground mine fire scenarios In underground mines, a fire can occur wherever flammable material is found, but predicting it at all possible locations is practically impossible. So by analyzing this list of fire locations that have potential flammable materials, it is down to those places that have the highest risk of fire occurrence [10]. The process of conducting fire risk assessment is very straightforward and does not need to be considered in any further detail in this research. What is new in this study is the proposal of methodology for quickly and efficiently locating and generating fire scenarios ready for simulation on the basis of which optimal evacuation plans will be developed. To identify possible locations for fire scenarios in underground mines, different approaches can be used, such as [2, 9]: Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 • Analysis of production plans Developing effective evacuation plans in case of fire in underground mine is the most important and sometimes the only option for safe evacuation of all involved in the fire scenario. The wide range of possibilities in the process of improving the evacuation plans in case of fire has motivated many researchers to make new or to modify the existing methodologies or procedures for developing effective and Ji et al. [3] developed a visual model to simulate the evacuation process of miners to determine the evacuation time, exit flow rate, and evacuation path and show that simulation is effective technology to establish safe evacuation system. Chen et al. [4] developed 3D CFD model to reconstruct the laneway conveyor belt fire scenes under two ventilating conditions to investigate the influence of smoke movement on miner evacuation behaviors. Wang et al. [5] through example demonstrated the use of their proposed framework for human error risk analysis of coal mine emergency evacuation and also the method to evaluate the reliability of human safety barriers. Wu et al. [6] conducted emergency evacuation simulation and visualized analysis of underground mine water bursting disaster scene, to achieve the simulation of the dynamic process of individual or group behavior and to provide platform for rational evacuation under the situation of mine disaster. Adjiski et al. [7–9] completed many different manuscripts and projects in the field of simulation and modeling of fire scenarios and evacuation plans in under- To the authors' best knowledge and the extensive search of literature, a lack of methodologies and systems that focus on developing evacuation plans in case of fire in underground mines is shown. Due to the large number of factors from which the effective evacuation process depends, this field of research requires continuous upgrading to address all challenges and also to provide optimal evacuation routes that sometimes represent the only option for preventing loss This chapter is an extension and upgrade of the previously published works In underground mines, a fire can occur wherever flammable material is found, but predicting it at all possible locations is practically impossible. So by analyzing this list of fire locations that have potential flammable materials, it is down to those places that have the highest risk of fire occurrence [10]. The process of conducting fire risk assessment is very straightforward and does not need to be considered in What is new in this study is the proposal of methodology for quickly and efficiently locating and generating fire scenarios ready for simulation on the basis of To identify possible locations for fire scenarios in underground mines, different from the same author and hopefully will contribute to the process that will improve the methodologies and systems for optimal fire evacuations in under- 2. Methodology for developing underground mine fire scenarios optimal evacuation plans. Fire Safety and Management Awareness ground mines. of human lives. ground mines. any further detail in this research. approaches can be used, such as [2, 9]: • Fire risk assessment 72 which optimal evacuation plans will be developed. • Historical records of fire incidents in the mine • Analysis of work processes and mechanization, etc. The dynamics of mining activities to increase and fulfill production capacity generates a constant shift in production sites generally associated with mechanization that is likely to trigger a fire scenario. Due to this fact as a relevant indicator that realistically reflects and constantly updates, the list of possible fire locations would be a detailed analysis of daily or monthly production plans. This step involves a thorough analysis of the daily/monthly production plans that will detect any flammable materials mostly associated with the mechanization needed to achieve the required production capacity. A case study of the "SASA"-R.N. Macedonia mine was used in order to conduct the necessary steps presented on Figure 1. The steps shown in Figure 1 are based on a simple analysis of the production plans that can detect all workplaces with the appropriate work cycle together with the related mechanization which is often associated with fire scenarios. To demonstrate the presented methodology, a 3D model of the underground ventilation network of the mine "SASA"-R.N. Macedonia is prepared on which all the necessary analysis and simulations will be performed (Figure 2). On the ventilation map, the possible fire locations along with the group of mineworkers identified using the proposed methodology on Figure 1 and also the possible exits from the underground mine are also marked. The process of modeling fire scenarios is closely related to the degree of uncertainty when it comes to the input data, which largely depends on the size of the fire itself [11, 12]. Examples of such input parameters that affect the fire models in underground mines are fire load, fire location, burn rate of materials, heat release rate, ventilation parameters, etc. Due to the stochastic nature of the input parameters related to the fire models, the appropriate results should be treated with caution. From the large list of stochastic input parameters, the authors decided to elaborate only on the process of obtaining fire load inputs, which largely depends on Figure 2. Ventilation map of the "SASA" mine with marked possible fire locations, group of mineworkers, and exits. the severity of the fire scenario itself. The process of modeling fire load inputs that are closely related to the inability to accurately determine the type and quantity of flammable material covered by a fire scenario is done using the Monte Carlo simulation technique. The reason for selecting and analyzing the fire load parameter is because of its immense contribution in generating the amount of toxic gases from which the complexity of the evacuation process depends. The reason for choosing the Monte Carlo simulation technique is because of its speed and simplicity of implementation and also the ability to generate a large amount of input data sampled randomly from their respective distributions [13–15]. The process of developing this model that incorporates the Monte Carlo simulation technique associated with the normal distribution defined by mean = 50, and standard deviation = 15, has been previously explained by the same author, and the entire methodology and reasons for selecting the highlighted parameters can be found here [16]. The inputs in the next steps of the proposed methodology are the approximate values of the total fire load for the selected mechanization. To simplify the process of determining this data, we used the technical manual of the Scooptram ST7, from which we approximated the quantities for the tire, hydraulic fluid, and diesel fuel which will be threatened as total fire load (Table 1). Regardless of the fact that the amount of diesel fuel is stochastic in nature, and is dependent on a number of factors, to simplify the model, we will consider it a known value, and we will treat Generated scenarios along with the corresponding fire load distribution obtained from the Monte Carlo Tire [kg] Diesel fuel [L] Hydraulic fluid [L] Scooptram ST7 238 \* 4 (tires) = 952 190 111 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios Approximate fire load calculation for the fire scenario from Scooptram ST7. DOI: http://dx.doi.org/10.5772/intechopen.91213 Following the analysis of the approximate amount of fire load, the next step is to model them using the previously mentioned Monte Carlo simulation technique, along with the necessary data for its normal distribution defined by mean and For the purpose of this study using the Monte Carlo simulation model, we have generated 20 scenarios with different fire load distribution, which will give variations in the results from the fire scenarios, and we will analyze their impact on the The purpose of fire models is to describe fire characteristics, such as heat release Various case studies previously published from the same author are based on the modeling of fire scenarios in a number of different mine ventilation layouts [7–9]. 3. Modeling and simulation of fire scenarios in underground mines rate, the burning rate of material, smoke, generating toxic gases, etc., and the results of simulating these models will be as good as the inputs [9, 17]. In order to create a relevant fire model in underground mines, it must be based on an accurate ventilation model. This interconnection and accuracy of the fire and ventilation models will depend on the movement of smoke and toxic gases through the mine facilities from which the evacuation process is based. it in a further expansion of the research. standard deviation [16]. Table 1. Figure 3. 75 simulation model. evacuation process (Figure 3). What is new in this research is the development of a database that includes all fire scenarios in a predetermined location using the abovementioned methodology on Figure 1. All fire scenarios are analyzed in terms of impact from the fire load input parameters on the evacuation process, that is, how different distribution of combustible materials from the same mechanization (or other composition of combustible materials) will impact the evacuation process. The introduction of this database aims to select fire scenarios of the same type but with different fire load distribution, from which we can analyze the effects on the evacuation process. The results of this analysis can be used to improve the design of fire systems and evacuation plans and to test them for their effectiveness in different conditions. From the simple analysis of the monthly production plan of "SASA" mine, we have extracted all work sites for ore exploitation and development of mining facilities with the appropriate work cycle together with the related mechanization. To present the methodology, we will only analyze fire scenarios generated by only one mechanization and present the optimal evacuation route for only one group of workers. For the purposes of this analysis, we will present the results of the fire scenarios generated by the mechanization Scooptram ST7, located at the possible fire location 3, from where we will simulate the fire scenarios and calculate the optimal evacuation route for group 1 (Figure 2). Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Table 1. Approximate fire load calculation for the fire scenario from Scooptram ST7. #### Figure 3. the severity of the fire scenario itself. The process of modeling fire load inputs that are closely related to the inability to accurately determine the type and quantity of flammable material covered by a fire scenario is done using the Monte Carlo simulation technique. The reason for selecting and analyzing the fire load parameter is because of its immense contribution in generating the amount of toxic gases from which the complexity of the evacuation process depends. The reason for choosing the Monte Carlo simulation technique is because of its speed and simplicity of implementation and also the ability to generate a large amount of input data sampled randomly from their respective Ventilation map of the "SASA" mine with marked possible fire locations, group of mineworkers, and exits. The process of developing this model that incorporates the Monte Carlo simulation technique associated with the normal distribution defined by mean = 50, and standard deviation = 15, has been previously explained by the same author, and the entire methodology and reasons for selecting the highlighted parameters can be What is new in this research is the development of a database that includes all fire scenarios in a predetermined location using the abovementioned methodology The introduction of this database aims to select fire scenarios of the same type but with different fire load distribution, from which we can analyze the effects on the evacuation process. The results of this analysis can be used to improve the design of fire systems and evacuation plans and to test them for their effectiveness From the simple analysis of the monthly production plan of "SASA" mine, we have extracted all work sites for ore exploitation and development of mining facilities with the appropriate work cycle together with the related mechanization. To present the methodology, we will only analyze fire scenarios generated by only one mechanization and present the optimal evacuation route for only one group of For the purposes of this analysis, we will present the results of the fire scenarios generated by the mechanization Scooptram ST7, located at the possible fire location 3, from where we will simulate the fire scenarios and calculate the optimal All fire scenarios are analyzed in terms of impact from the fire load input parameters on the evacuation process, that is, how different distribution of com- bustible materials from the same mechanization (or other composition of combustible materials) will impact the evacuation process. distributions [13–15]. Fire Safety and Management Awareness found here [16]. in different conditions. evacuation route for group 1 (Figure 2). on Figure 1. Figure 2. workers. 74 Generated scenarios along with the corresponding fire load distribution obtained from the Monte Carlo simulation model. The inputs in the next steps of the proposed methodology are the approximate values of the total fire load for the selected mechanization. To simplify the process of determining this data, we used the technical manual of the Scooptram ST7, from which we approximated the quantities for the tire, hydraulic fluid, and diesel fuel which will be threatened as total fire load (Table 1). Regardless of the fact that the amount of diesel fuel is stochastic in nature, and is dependent on a number of factors, to simplify the model, we will consider it a known value, and we will treat it in a further expansion of the research. Following the analysis of the approximate amount of fire load, the next step is to model them using the previously mentioned Monte Carlo simulation technique, along with the necessary data for its normal distribution defined by mean and standard deviation [16]. For the purpose of this study using the Monte Carlo simulation model, we have generated 20 scenarios with different fire load distribution, which will give variations in the results from the fire scenarios, and we will analyze their impact on the evacuation process (Figure 3). #### 3. Modeling and simulation of fire scenarios in underground mines The purpose of fire models is to describe fire characteristics, such as heat release rate, the burning rate of material, smoke, generating toxic gases, etc., and the results of simulating these models will be as good as the inputs [9, 17]. In order to create a relevant fire model in underground mines, it must be based on an accurate ventilation model. This interconnection and accuracy of the fire and ventilation models will depend on the movement of smoke and toxic gases through the mine facilities from which the evacuation process is based. Various case studies previously published from the same author are based on the modeling of fire scenarios in a number of different mine ventilation layouts [7–9]. For this study, i.e., for simulating fire models across the 3D ventilation network, we used the VentSim software along with VentFIRE™ module that are interconnected because they belong to the same software package. With the help of VentSim software a 3D ventilation network with all working parameters is developed, while the VentFIRE™ module is used for simulation and calculation of the fire scenarios previously generated with the Monte Carlo simulation model. The theoretical and the working principle of the VentSim software together with the VentFIRE™ module can be found here [18]. Fire models in some cases are analyzed by CFD software for the purpose of comparison between the results obtained from simpler computational methods. Due to the size and complexity of the underground mines, it should be emphasized that CFD analysis can only be used to represent a small section of the mine. The results of such CFD analyses that require a large number of computations which will generate only results related to the immediate proximity of the fire scenario cannot realistically represent the full image generated by the fire model [8, 19]. The functionality of the methodology presented in this chapter is based on the modeling and simulation of fire scenarios whose results can fully represent each time interval of the movement of smoke and fire gases through the whole ventilation network from which the evacuation process entirely depends. In the process of modeling fire scenarios in VentFIRE™ module in addition to the fire load data presented in Figure 3, which was generated with the Monte Carlo simulation model, specific data are also required for each material which is presented in Table 2. For the purpose of providing this data, laboratory tests or fire databases containing such information may be used [20, 21]. The results of the fire models obtained by the VentFIRE™ module are in the form of a dynamic representation of the real-time fire progression and utilize a graphic visualization of the spread and concentration of combustion products and all the fire-related data throughout the ventilation system (Figure 4). Monitoring points that are strategically placed throughout the ventilation network allow the extraction of data in the form of concentrations over time for all fire-related data. In this study, for the evaluation of the evacuation plans, only the CO concentration over time curve will be analyzed throughout the ventilation network. The results from the monitoring points will serve for realistic mapping of the CO inhalation throughout the evacuation route for anyone affected by the fire scenario. Figure 5 shows the CO concentration measured from the monitoring point at the location for the fire scenario S-1. Figure 6 shows the average values of CO concentration vs. total duration time Average values of CO concentration at fire location and total time duration of the fire for all scenarios generated for all fire scenario variants generated by the Monte Carlo simulation model, measured from the fire location. by the Monte Carlo simulation model. Figure 4. Figure 5. Figure 6. 77 Screenshot from the fire scenario S-1 at 30 minutes from the fire ignition. DOI: http://dx.doi.org/10.5772/intechopen.91213 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios CO concentration over time curve at the fire scenario S-1 location. #### Table 2. Input fire characteristics data for the fire load. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Figure 4. For this study, i.e., for simulating fire models across the 3D ventilation network, The theoretical and the working principle of the VentSim software together with In the process of modeling fire scenarios in VentFIRE™ module in addition to the fire load data presented in Figure 3, which was generated with the Monte Carlo presented in Table 2. For the purpose of providing this data, laboratory tests or fire The results of the fire models obtained by the VentFIRE™ module are in the form of a dynamic representation of the real-time fire progression and utilize a graphic visualization of the spread and concentration of combustion products and Monitoring points that are strategically placed throughout the ventilation network allow the extraction of data in the form of concentrations over time for all fire-related data. In this study, for the evaluation of the evacuation plans, only the CO concentration over time curve will be analyzed throughout the ventilation network. The results from the monitoring points will serve for realistic mapping of the CO inhalation throughout the evacuation route for anyone affected by the fire scenario. Figure 5 shows the CO concentration measured from the monitoring point ] 1150 832 760 Simplified chemical hydrocarbon formula C4H6 C12H23 C36H74 Heat of combustion [MJ/kg] 44 45 48 Burning rate of material [kg/m<sup>2</sup> ∗ s] 0.062 0.045 0.039 O2 consumed [kg/kg] 3.62 3.33 3.57 Yield CO2 [kg/kg] 0.9 3.2 3.3 Yield CO min [kg/kg] 0.13 0.019 0.1 Yield CO max [kg/kg] 0.23 0.21 0.24 Yield soot [kg/kg] 0.1 0.059 0.1 Tire Diesel fuel Hydraulic fluid simulation model, specific data are also required for each material which is all the fire-related data throughout the ventilation system (Figure 4). databases containing such information may be used [20, 21]. at the location for the fire scenario S-1. Input fire characteristics data for the fire load. the VentFIRE™ module can be found here [18]. Fire models in some cases are analyzed by CFD software for the purpose of comparison between the results obtained from simpler computational methods. Due to the size and complexity of the underground mines, it should be emphasized that CFD analysis can only be used to represent a small section of the mine. The results of such CFD analyses that require a large number of computations which will generate only results related to the immediate proximity of the fire scenario cannot realistically represent the full image generated by the fire model [8, 19]. The functionality of the methodology presented in this chapter is based on the modeling and simulation of fire scenarios whose results can fully represent each time interval of the movement of smoke and fire gases through the whole ventilation network from which the evacuation process interconnected because they belong to the same software package. With the help of VentSim software a 3D ventilation network with all working parameters is developed, while the VentFIRE™ module is used for simulation and calculation of the fire scenarios previously generated with the Monte Carlo simulation model. we used the VentSim software along with VentFIRE™ module that are entirely depends. Fire Safety and Management Awareness Density [kg/m<sup>3</sup> Table 2. 76 Figure 5. CO concentration over time curve at the fire scenario S-1 location. #### Figure 6. Average values of CO concentration at fire location and total time duration of the fire for all scenarios generated by the Monte Carlo simulation model. Figure 6 shows the average values of CO concentration vs. total duration time for all fire scenario variants generated by the Monte Carlo simulation model, measured from the fire location. These results highlight the impact of different fire load distribution, thus providing additional data for analysis during the process for determining the optimal evacuation routes. #### 4. Methodology for determining the optimal evacuation routes based on simulated fire scenarios #### 4.1 Life safety assessment during evacuation based on fractional effective dose (FED) from CO inhalation Statistical underground mine fire evidence shows that most injuries and deaths are not caused by direct contact with the fire but by way of smoke and toxic gases inhalation [22]. While the fire scenario may be confined to a localized underground mine area, the smoke produced will rise and with the help of the ventilation system may spread rapidly through the mine. The spread of smoke and toxic gases through the underground mine network will cause difficulties in the evacuation process, and therefore, there is a need for an effective methodology for planning and developing of optimal evacuation routes. Purser [23] gives extensive review of smoke and toxic gases hazards, including exposure thresholds that can cause incapacitation and even death. In underground mine fires, the most common asphyxiate is CO, and its effects of incapacitation depend from the gas concentrations and the durations of exposure. The evacuation management system must be designed and evaluated against a set of criteria to ensure safe evacuation of the mineworkers, which can be achieved by analyzing the fire environments using modeling and simulation. The proposed method in this book chapter involves the determination of accumulating exposure effect at regular discrete time increments to get the cumulative dosage in terms of FED for the total period of exposure. The exposure doses are calculated as a fraction of incapacitation at every time increment, and the value of FED = 1.0 represents the state of incapacitation in which mineworkers are incapable of completing their own evacuation. Purser [24] suggests mathematical model for estimating toxic hazard from inhalation of CO from fire scenario in terms of time to incapacitation or death in form of FED and is given as follows: $$\text{FED}\_{\text{Taxicity}} = \text{FED}\_{\text{CO}} \* \text{V}\_{\text{CO}\_2} + \text{FED}\_{\text{O}\_2} \tag{1}$$ $$\text{FED}\_{\text{CO}} = \sum\_{\text{t}\_1}^{\text{t}\_2} \frac{\text{K} \ast [\text{CO}]^{1,036}}{\text{D}} \Delta \text{t} \tag{2}$$ One of the limitations of this model is the lack of a clear safety margin between the values of the FED in which the transition in the evacuation process from safe to unsafe zone begins. As previously stated, for the evacuation to be considered safe, Activity K D At rest 2,81945 ∗ 10<sup>4</sup> 40 Light work 8,2925 ∗ 10<sup>4</sup> 30 Heavy work 1,6585 ∗ 10<sup>4</sup> 20 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios 5–10 Shortness of breath on vigorous exertion, possible tightness across forehead, statistically significant diminution of visual perception, manual dexterity, or ability to learn 21–30 Severe headache, pulsation in sides of head, impaired thinking, disturbed vision, fainting, 41–50 Brain damage, lethargy, seizures, syncope, death from severe cellular hypoxia if exposure 11–20 Atypical dyspnea, throbbing headache, dizziness, nausea, confusion and decreased 31–40 Severe headache, dizziness, respiratory failure, coma, intermittent convulsions To improve the methodology in this regard, additional model is introduced that will allow to link the entire evacuation timeline with another parameter in the form of COHb prediction in the blood as a result of the CO inhalation generated by the 4.2 Model for predicting carboxyhemoglobin (COHb) concentration as a result The overwhelming hazard in fires is the COHb buildup in the blood as a result of exposures to CO. Inhaled CO acts on the human body by competing with oxygen to combine with hemoglobin molecules in the blood, forming COHb rather than normal oxyhemoglobin (O2Hb) [25]. Exposure to a large concentration of CO is lethal, and the signs and symptoms produced are directly related to the percentage of The most widely used mathematical model (Coburn-Forster-Kane (CFK)) was implemented in order to predict COHb (%) blood level from CO exposure on mineworkers during the underground mine fire scenario. the FED value should be <1. The question here is how much less than 1? fire scenario. 79 Table 4. Table 3. Values for different activity levels for the constants K and D. 2,5–4 Decreased exercise performance in patients with angina exercise tolerance, dilatation of skin vessels 51–60 Same as above, coma, convulsions, Cheyne-Stokes respiration >70 Slowing and stopping of respiration and death within short period Approximate clinical symptoms associated with the blood COHb (%) level [26]. easy fatigability, disturbed judgment COHb (%) Clinical symptoms 0,4–1 Normal value for nonsmokers DOI: http://dx.doi.org/10.5772/intechopen.91213 is prolonged of CO inhalation COHb in the blood (Table 4). $$V\_{\rm CO\_2} = \frac{\exp\left(0, 1903 \ast \text{\%CO2} + 2, 0004\right)}{7, 1} \tag{3}$$ $$\text{FED}\_{\text{O}\_2} = \sum\_{\text{t}\_1}^{\text{t}\_2} \frac{1}{\exp\left(8, 13 - 0, 54\left(20, 9\% - 9\% O\_2\right)\right)} \Delta \mathbf{t} \tag{4}$$ where CO (carbon monoxide) is the average concentration (ppm) over the time increment Δt in minutes, K and D are constants which depend on the activity of the person (Table 3), %CO2 is the carbon dioxide concentration, and (20,9-%O2) is the oxygen vitiation over the time increment Δt. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Table 3. These results highlight the impact of different fire load distribution, thus providing additional data for analysis during the process for determining the optimal 4. Methodology for determining the optimal evacuation routes based 4.1 Life safety assessment during evacuation based on fractional effective dose Statistical underground mine fire evidence shows that most injuries and deaths are not caused by direct contact with the fire but by way of smoke and toxic gases While the fire scenario may be confined to a localized underground mine area, the smoke produced will rise and with the help of the ventilation system may spread The spread of smoke and toxic gases through the underground mine network will cause difficulties in the evacuation process, and therefore, there is a need for an effective methodology for planning and developing of optimal evacuation routes. Purser [23] gives extensive review of smoke and toxic gases hazards, including In underground mine fires, the most common asphyxiate is CO, and its effects of incapacitation depend from the gas concentrations and the durations of exposure. The evacuation management system must be designed and evaluated against a set of criteria to ensure safe evacuation of the mineworkers, which can be achieved The proposed method in this book chapter involves the determination of accumulating exposure effect at regular discrete time increments to get the cumulative dosage in terms of FED for the total period of exposure. The exposure doses are calculated as a fraction of incapacitation at every time increment, and the value of FED = 1.0 represents the state of incapacitation in which mineworkers are incapable Purser [24] suggests mathematical model for estimating toxic hazard from inhalation of CO from fire scenario in terms of time to incapacitation or death in t1 <sup>V</sup>CO2 <sup>¼</sup> exp 0, 1903 ð Þ <sup>∗</sup> %CO2 <sup>þ</sup> 2, 0004 where CO (carbon monoxide) is the average concentration (ppm) over the time increment Δt in minutes, K and D are constants which depend on the activity of the person (Table 3), %CO2 is the carbon dioxide concentration, and (20,9-%O2) is FEDToxicity ¼ FEDCO ∗ VCO2 þ FEDO2 (1) Δt (2) Δt (4) 7, 1 (3) <sup>K</sup><sup>∗</sup> ½ � CO 1,036 D 1 exp 8, 13 ð Þ � 0, 54 20, 9% ð Þ � %O<sup>2</sup> exposure thresholds that can cause incapacitation and even death. by analyzing the fire environments using modeling and simulation. FEDCO <sup>¼</sup> <sup>X</sup>t2 evacuation routes. inhalation [22]. rapidly through the mine. on simulated fire scenarios Fire Safety and Management Awareness (FED) from CO inhalation of completing their own evacuation. form of FED and is given as follows: FEDO2 <sup>¼</sup> <sup>X</sup>t2 the oxygen vitiation over the time increment Δt. 78 t1 Values for different activity levels for the constants K and D. #### Table 4. Approximate clinical symptoms associated with the blood COHb (%) level [26]. One of the limitations of this model is the lack of a clear safety margin between the values of the FED in which the transition in the evacuation process from safe to unsafe zone begins. As previously stated, for the evacuation to be considered safe, the FED value should be <1. The question here is how much less than 1? To improve the methodology in this regard, additional model is introduced that will allow to link the entire evacuation timeline with another parameter in the form of COHb prediction in the blood as a result of the CO inhalation generated by the fire scenario. #### 4.2 Model for predicting carboxyhemoglobin (COHb) concentration as a result of CO inhalation The overwhelming hazard in fires is the COHb buildup in the blood as a result of exposures to CO. Inhaled CO acts on the human body by competing with oxygen to combine with hemoglobin molecules in the blood, forming COHb rather than normal oxyhemoglobin (O2Hb) [25]. Exposure to a large concentration of CO is lethal, and the signs and symptoms produced are directly related to the percentage of COHb in the blood (Table 4). The most widely used mathematical model (Coburn-Forster-Kane (CFK)) was implemented in order to predict COHb (%) blood level from CO exposure on mineworkers during the underground mine fire scenario. Previous research by several authors validated both linear and nonlinear CFK model against observations made on subjects exposed to variable CO concentrations, and the consensus is that the model predictions works quite well. The CFK nonlinear model is given by the following Equation [27]: $$\left[\text{COHb}\right]\_{\text{t}} = \frac{1}{\mathbf{A}\left(\frac{\text{AC}}{[\text{COHb}]\_{\text{0}}}\right)} + (\mathbf{1} - \mathbf{C})\mathbf{V}\_{\text{CO}}\mathbf{B} + (\mathbf{1} - \mathbf{C})\mathbf{P}\_{\text{1,CO}}\tag{5}$$ $$\mathbf{A} = \frac{\mathbf{PO}\_2}{\mathbf{M}[\mathbf{O}\_2 \mathbf{H} \mathbf{b}]} \tag{6}$$ $$\mathbf{B} = \frac{\mathbf{1}}{\mathbf{D}} + \frac{\mathbf{P}}{\mathbf{V\_a}} \tag{7}$$ $$\mathbf{C} = \mathbf{e}^{(-\frac{\mathbf{t} \cdot \mathbf{t}}{\nabla\_{\mathbf{b}} \mathbf{B}})} \tag{8}$$ These factors that influence mineworkers' escape speed can increase the exposure time from the fire scenario and thus present very important factors to be Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios We defined the mineworkers' normal evacuation speed by v0, and under the The tunnel slope influences the mineworkers' evacuation (and also walking) speed, and the greater the slope the more influence it will have on the process. The tunnel slope influence under climbing situation is given by the following where m is the standard human mass [kg], g is the gravity acceleration [m/s<sup>2</sup> The smoke generated by the fire scenario is a major factor in determining tunnel visibility. This visibility factor has important effects on the evacuation speed of Based on the reviewed literature, two threshold values hold a central function during an evacuation in a smoke-filled environment [30, 32]. The first threshold value is the visibility level at which evacuees in general can be expected to start reducing their evacuation speed. This value based on the reviewed experiments of the data presented from the literature was set to 3 meters as corresponding visibility The second threshold value is the visibility level at which the mineworkers can Practically, in the process of calculating the reduction of evacuation speed based • All individuals in the group are assumed to be evacuating with the same speed. • Visibility levels >3 m: mineworkers' evacuation speed is represented by be assumed to be evacuating with their slowest speed. Based on the reviewed literature, the slowest speed during an evacuation in a smoke-filled environment is similar to movement in complete darkness which can be expected to be about 0,2 m/s [30]. In this analysis, the value for the slowest speed of evacuation will also be applied when the mineworkers will move through the evacuation stairs in the on the smoke visibility level, the model is set in the following way: When mineworkers pass down slope tunnels, we will assume no influence on their speed, and the model will treat this as normal evacuation speed v0 þ cos θ<sup>s</sup> (9) ], kts <sup>¼</sup> mgv0 sin <sup>θ</sup><sup>s</sup> P0 considered in the process of determining optimal evacuation routes. influence of the above factors, the evacuation speed will be vf. Methodology for implementation of the evacuation speed influence model. DOI: http://dx.doi.org/10.5772/intechopen.91213 and θ<sup>s</sup> is the tunnel's angle of slope in degrees. the mineworkers who are escaping. threshold value [30, 33, 34]. ventilation raise. 1,2 m/s 81 Equation [31]: Figure 7. (i.e., kts ¼ 1Þ: where: M—Haldane constant, ratio of the affinity of Hb for CO to that of O2 = 240. ½ � O2Hb —oxyhemoglobin concentration = 0,2 ml ml�<sup>1</sup> blood. ½ � COHb <sup>t</sup>—carboxyhemoglobin concentration at time t in ml CO per ml blood. ½ � COHb <sup>0</sup>—initial concentration of carboxyhaemoglobin in blood (%COHb = 0,5% for nonsmokers; %COHb >2% for 80% of smokers; %COHb = 10% for heavy smokers). PO2—partial pressure of oxygen in lung capillaries = 13,3 kPa. VCO—endogenous CO production rate = 0,007 ml min�<sup>1</sup> . D—diffusion capacity of the lungs for CO = 225 ml min�<sup>1</sup> kPa (in reality this is not a constant but is altered by a number of factors including exercise). P—Barometric pressure - saturated vapor pressure of water at 37°C = 95,1 kPa. Vb—blood volume 5500 ml. P1,CO—partial pressure of CO in inspired air = 0,0101 kPa (adopted for the purposes of this model). Va—alveolar ventilation rate = 6000 ml min�<sup>1</sup> . t—duration of exposure [min]. The limitations in the CFK model are located with the physiological variables needed as input to the model which are difficult to measure, such as blood volume, endogenous production of CO, and the pulmonary diffusing capacity [28]. For the purpose of this study, an Excel model based on the CFK equation is built to predict the individual's COHb formation (%), as a result from CO inhalation. For simplification purposes the abovementioned physiological variables are set as default values (as defined in the equation). The proposed model for predicting COHb (%) with appropriate clinical symptoms (Table 4) connected with the FED model can better determine the threshold in which the evacuation will be considered safe. #### 4.3 Model for the conversion of the factors that influence the speed of evacuation To be able to calculate the optimal evacuation routes in underground mines, details about the tunnels' parameters should be provided. Each fire scenario generates factors that influence the complexity and the speed of the evacuation itself. Based on extensive literature review, two factors are located that have most influence on the evacuation speed, and these factors are generalized in the form of tunnel slope and smoke visibility [7, 29–31]. The model framework is shown in Figure 7. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Figure 7. Previous research by several authors validated both linear and nonlinear CFK model against observations made on subjects exposed to variable CO concentrations, and the consensus is that the model predictions works quite well. The CFK <sup>A</sup> <sup>¼</sup> PO2 P Va ð� tA <sup>B</sup> <sup>¼</sup> <sup>1</sup> D þ C ¼ e ½ � O2Hb —oxyhemoglobin concentration = 0,2 ml ml�<sup>1</sup> blood. ½ � COHb <sup>0</sup>—initial concentration of carboxyhaemoglobin in blood PO2—partial pressure of oxygen in lung capillaries = 13,3 kPa. VCO—endogenous CO production rate = 0,007 ml min�<sup>1</sup> not a constant but is altered by a number of factors including exercise). M—Haldane constant, ratio of the affinity of Hb for CO to that of O2 = 240. ½ � COHb <sup>t</sup>—carboxyhemoglobin concentration at time t in ml CO per ml blood. (%COHb = 0,5% for nonsmokers; %COHb >2% for 80% of smokers; %COHb = 10% D—diffusion capacity of the lungs for CO = 225 ml min�<sup>1</sup> kPa (in reality this is P—Barometric pressure - saturated vapor pressure of water at 37°C = 95,1 kPa. . P1,CO—partial pressure of CO in inspired air = 0,0101 kPa (adopted for the The limitations in the CFK model are located with the physiological variables needed as input to the model which are difficult to measure, such as blood volume, For the purpose of this study, an Excel model based on the CFK equation is built to predict the individual's COHb formation (%), as a result from CO inhalation. For simplification purposes the abovementioned physiological variables are set as The proposed model for predicting COHb (%) with appropriate clinical symptoms (Table 4) connected with the FED model can better determine the threshold To be able to calculate the optimal evacuation routes in underground mines, details about the tunnels' parameters should be provided. Each fire scenario generates factors that influence the complexity and the speed of the evacuation itself. Based on extensive literature review, two factors are located that have most influence on the evacuation speed, and these factors are generalized in the form of tunnel slope and smoke visibility [7, 29–31]. The model framework is shown in endogenous production of CO, and the pulmonary diffusing capacity [28]. 4.3 Model for the conversion of the factors that influence the speed of <sup>þ</sup> ð Þ <sup>1</sup> � <sup>C</sup> VCOB <sup>þ</sup> ð Þ <sup>1</sup> � <sup>C</sup> P1,CO (5) M O½ � 2Hb (6) VbB<sup>Þ</sup> (8) . (7) nonlinear model is given by the following Equation [27]: A AC ½ � COHb <sup>0</sup> ½ � COHb <sup>t</sup> <sup>¼</sup> <sup>1</sup> Fire Safety and Management Awareness where: for heavy smokers). Vb—blood volume 5500 ml. t—duration of exposure [min]. default values (as defined in the equation). in which the evacuation will be considered safe. Va—alveolar ventilation rate = 6000 ml min�<sup>1</sup> purposes of this model). evacuation Figure 7. 80 Methodology for implementation of the evacuation speed influence model. These factors that influence mineworkers' escape speed can increase the exposure time from the fire scenario and thus present very important factors to be considered in the process of determining optimal evacuation routes. We defined the mineworkers' normal evacuation speed by v0, and under the influence of the above factors, the evacuation speed will be vf. The tunnel slope influences the mineworkers' evacuation (and also walking) speed, and the greater the slope the more influence it will have on the process. The tunnel slope influence under climbing situation is given by the following Equation [31]: $$\mathbf{k}\_{\rm ts} = \frac{\text{mg} \mathbf{v}\_0 \sin \theta\_\mathbf{s}}{\mathbf{P}\_0} + \cos \theta\_\mathbf{s} \tag{9}$$ where m is the standard human mass [kg], g is the gravity acceleration [m/s<sup>2</sup> ], and θ<sup>s</sup> is the tunnel's angle of slope in degrees. When mineworkers pass down slope tunnels, we will assume no influence on their speed, and the model will treat this as normal evacuation speed v0 (i.e., kts ¼ 1Þ: The smoke generated by the fire scenario is a major factor in determining tunnel visibility. This visibility factor has important effects on the evacuation speed of the mineworkers who are escaping. Based on the reviewed literature, two threshold values hold a central function during an evacuation in a smoke-filled environment [30, 32]. The first threshold value is the visibility level at which evacuees in general can be expected to start reducing their evacuation speed. This value based on the reviewed experiments of the data presented from the literature was set to 3 meters as corresponding visibility threshold value [30, 33, 34]. The second threshold value is the visibility level at which the mineworkers can be assumed to be evacuating with their slowest speed. Based on the reviewed literature, the slowest speed during an evacuation in a smoke-filled environment is similar to movement in complete darkness which can be expected to be about 0,2 m/s [30]. In this analysis, the value for the slowest speed of evacuation will also be applied when the mineworkers will move through the evacuation stairs in the ventilation raise. Practically, in the process of calculating the reduction of evacuation speed based on the smoke visibility level, the model is set in the following way: Figure 8. Representation of relative reduction of speed in a smoke-filled environment according to the model. • Visibility levels ≤3 m: mineworkers' evacuation speed is represented by a relative reduction of 0,34 m/s per meter visibility in a smoke-filled environment down to the previously defined minimum speed of 0,2 m/s. The correlation in this model is described by the following equation and by Figure 8 [30]: $$w = \min\left(1; \max\left(0, 2; 1, 2 - 0, 34 \* (3 - V)\right)\right) \tag{10}$$ where w is the evacuation speed [m/s] and V the visibility [m]. #### 5. Results and discussion Determining the optimal routes for evacuation in the case of underground mine fire makes the difference between life and death. In this book chapter, we established a methodology for calculating the optimal routes for evacuation in case of underground mine fire based on simulated scenarios. The methodology shown in Figure 9 provides the necessary steps to assess the potential fire scenarios and to generate the necessary data on the basis of which all evacuation routes will be evaluated and the optimization process implemented. The methodology consists of three parts, i.e., developing underground mine fire scenarios, modeling and simulation of fire scenarios, and determining the optimal evacuation routes based on the generated results. The parts of the presented methodology and the procedures for their implementation are presented in detail above. For the purpose of this study, a case study of the "SASA"-R.N. Macedonia mine was used for determining the optimal routes for evacuations. In the process of calculating all the parameters needed to determine the optimal evacuation routes, we will take into account the self-contained self-rescuer (SCSR). The use of SCSR in underground mining is a legal obligation in almost all countries around the world, so its introduction into the process of determining the optimal evacuation routes is a very important factor. The SCSR is a portable device that is used in underground mines to provide breathable air for the mineworkers when the surrounding atmosphere is filed with contaminants after emergency situation. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Figure 9. 83 Proposed methodology implementation framework. Extensive research on fire reports provides the fact that sometimes this first line of defense from smoke inhalation in the form of SCSR fails to function properly due to technical problems or due to insufficient training of the mineworkers [35]. Because of this fact in this study, we will make two parallel analyses to calculate the To present all the steps that the methodology is consists of, we will present the results obtained from only one fire location from which we will calculate the optimal evacuation routes for only one group of workers for all of the 20 fire scenarios generated by the Monte Carlo simulation model. The results from the Monte Carlo simulation (Figure 3) are used as input fire load data for modeling and simulating fire scenarios in the VentFIRE™ module through the mine ventilation network (Figure 2). Following the simulation of all 20 fire scenarios from the same fire location, all possible evacuation routes for group 1 have been identified (Figure 10). Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Figure 9. Proposed methodology implementation framework. In the process of calculating all the parameters needed to determine the optimal evacuation routes, we will take into account the self-contained self-rescuer (SCSR). The use of SCSR in underground mining is a legal obligation in almost all countries around the world, so its introduction into the process of determining the optimal evacuation routes is a very important factor. The SCSR is a portable device that is used in underground mines to provide breathable air for the mineworkers when the surrounding atmosphere is filed with contaminants after emergency situation. Extensive research on fire reports provides the fact that sometimes this first line of defense from smoke inhalation in the form of SCSR fails to function properly due to technical problems or due to insufficient training of the mineworkers [35]. Because of this fact in this study, we will make two parallel analyses to calculate the • Visibility levels ≤3 m: mineworkers' evacuation speed is represented by a relative reduction of 0,34 m/s per meter visibility in a smoke-filled environment down to the previously defined minimum speed of 0,2 m/s. Representation of relative reduction of speed in a smoke-filled environment according to the model. The correlation in this model is described by the following equation and by Determining the optimal routes for evacuation in the case of underground mine The methodology consists of three parts, i.e., developing underground mine fire scenarios, modeling and simulation of fire scenarios, and determining the optimal evacuation routes based on the generated results. The parts of the presented methodology and the procedures for their implementation are presented in detail above. For the purpose of this study, a case study of the "SASA"-R.N. Macedonia mine To present all the steps that the methodology is consists of, we will present the The results from the Monte Carlo simulation (Figure 3) are used as input fire load data for modeling and simulating fire scenarios in the VentFIRE™ module through the mine ventilation network (Figure 2). Following the simulation of all 20 fire scenarios from the same fire location, all possible evacuation routes for group 1 results obtained from only one fire location from which we will calculate the optimal evacuation routes for only one group of workers for all of the 20 fire established a methodology for calculating the optimal routes for evacuation in case of underground mine fire based on simulated scenarios. The methodology shown in Figure 9 provides the necessary steps to assess the potential fire scenarios and to generate the necessary data on the basis of which all evacuation routes will be where w is the evacuation speed [m/s] and V the visibility [m]. fire makes the difference between life and death. In this book chapter, we evaluated and the optimization process implemented. was used for determining the optimal routes for evacuations. scenarios generated by the Monte Carlo simulation model. have been identified (Figure 10). 82 w ¼ min 1; max 0, 2; 1, 2 ð Þ ð Þ � 0, 34 ∗ ð Þ 3 � V (10) Figure 8 [30]: Figure 8. 5. Results and discussion Fire Safety and Management Awareness Figure 10. Identification of possible evacuation routes for group 1 for all generated fire scenarios. #### Table 5. Results for group 1, evacuated along route 1 for scenario S-1. optimal evacuation routes in which we will introduce the use of a SCSR with a capacity of 30 minutes and the possibility of its non-functionality. By introducing this parameter in the form of functionality and non-functionality of SCSR, we can provide a detailed analysis that can predict the evacuation routes under different conditions. To elaborate on the proposed methodology, we will present in details the results of scenario S-1. After the development of the underground mine fire scenarios and their modeling and simulation inside the VentFIRE™ module, all the necessary data for the optimization process is gathered. For the purpose of this analysis, an average evacuation speed of 1,2 m/s is assumed. The average evacuation speed will be affected by the tunnel slope and smoke visibility. To calculate the impact on the average speed inside the evacuation process, An Excel model was built based on Eqs. 9 and 10. The results from the simulated fire scenario S-1, which are required as inputs for the FED, COHb, and route calculation models, are shown in Tables 5–8. All of the gathered results from the models are stored and arranged in the database. The next step of the proposed methodology is to filter the results inside the database through a route calculation model that will sort out all the evacuation routes according to the level of CO exposure, i.e., the results obtained from the FED The purpose of the route calculation model is to generate a list of all evacuation routes, which will include the data for route length and cumulative CO exposure in and COHb model. Position Section Position Section Position Section length [m] length [m] Table 6. Table 7. Table 8. 85 length [m] Visibility [m] DOI: http://dx.doi.org/10.5772/intechopen.91213 Results for group 1, evacuated along route 2 for scenario S-1. Visibility [m] Results for group 1, evacuated along route 3 for scenario S-1. Visibility [m] Results for group 1, evacuated along route 4 for scenario S-1. Slope [ o ] Slope [ o ] Slope [ o ] Reduction of evacuation speed (from visibility and slope) [m/s] Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios Reduction of evacuation speed (from visibility and slope) [m/s] Reduction of evacuation speed (from visibility and slope) [m/s] P1-P2 461 5,1 0 1,2 448 384 384 P2-P3 80 14 75 0,2 344 400 784 P3-P4 426 22 0 1,2 0 355 1139 P4-P5 671 25 0 1,2 0 559 1698 P5-P6 1689 25 0 1,2 0 1408 3106 P1-P2 667 5 0 1,2 448 556 556 P2-P3 340 2 6 0,34 881 1000 1556 P3-P4 80 25 75 0,2 0 400 1956 P4-P5 462 25 0 1,2 0 385 2341 P5-P6 1689 25 0 1,2 0 1408 3748 P1-P2 347 5,1 0 1,2 448 289 289 P2-P3 80 5,3 75 0,2 450 400 689 P3-P4 135 4,7 1,4 1 524 135 824 P4-P5 524 12 5,71 0,29 480 1807 2631 P5-P6 199 25 5,8 0,55 0 362 2993 P6-P7 797 25 1 0,65 0 1226 4219 Average CO (ppm) Average CO (ppm) Average CO (ppm) Evacuation time in section [s] Evacuation time in section [s] Evacuation time in section [s] Cumulative time [s] Cumulative time [s] Cumulative time [s] the form of a FED through the evacuation process. In the calculation process for the CO exposure over the entire evacuation route, we will include the SCSR in its two previously mentioned forms. To calculate the exposure from CO for each of the possible evacuation routes, the results shown in Tables 5–8 are used as inputs to the FED and the COHb model. The results from the CO exposure based on FED and COHb models build inside Excel are shown in Figures 11–14. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 #### Table 6. Results for group 1, evacuated along route 2 for scenario S-1. Table 7. optimal evacuation routes in which we will introduce the use of a SCSR with a capacity of 30 minutes and the possibility of its non-functionality. By introducing this parameter in the form of functionality and non-functionality of SCSR, we can provide a detailed analysis that can predict the evacuation routes under different Reduction of evacuation speed (from visibility and slope) [m/s] P1-P2 667 5 0 1,2 448 556 556 P2-P3 232 2 6 0,4 881 580 1136 P3-P4 495 4,6 6,1 0,3 514 1650 2786 P4-P5 524 12 5,71 0,29 480 1807 4593 P5-P6 199 25 5,8 0,55 0 362 4955 P6-P7 792 25 1 0,66 0 1200 6155 Average CO (ppm) Evacuation time in section [s] Cumulative time [s] Identification of possible evacuation routes for group 1 for all generated fire scenarios. Slope [ o ] To elaborate on the proposed methodology, we will present in details the results After the development of the underground mine fire scenarios and their modeling and simulation inside the VentFIRE™ module, all the necessary data for the For the purpose of this analysis, an average evacuation speed of 1,2 m/s is assumed. The average evacuation speed will be affected by the tunnel slope and smoke visibility. To calculate the impact on the average speed inside the evacuation process, An Excel model was built based on Eqs. 9 and 10. The results from the simulated fire scenario S-1, which are required as inputs for the FED, COHb, and route calculation In the calculation process for the CO exposure over the entire evacuation route, we will include the SCSR in its two previously mentioned forms. To calculate the exposure from CO for each of the possible evacuation routes, the results shown in Tables 5–8 are used as inputs to the FED and the COHb model. The results from the CO exposure based on FED and COHb models build inside Excel are shown in conditions. Table 5. Figure 10. Position Section length [m] Fire Safety and Management Awareness Visibility [m] Results for group 1, evacuated along route 1 for scenario S-1. of scenario S-1. Figures 11–14. 84 optimization process is gathered. models, are shown in Tables 5–8. Results for group 1, evacuated along route 3 for scenario S-1. Table 8. Results for group 1, evacuated along route 4 for scenario S-1. All of the gathered results from the models are stored and arranged in the database. The next step of the proposed methodology is to filter the results inside the database through a route calculation model that will sort out all the evacuation routes according to the level of CO exposure, i.e., the results obtained from the FED and COHb model. The purpose of the route calculation model is to generate a list of all evacuation routes, which will include the data for route length and cumulative CO exposure in the form of a FED through the evacuation process. The first step in the optimization model is to group the evacuation routes into Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios The values of the FED parameter on which the grouping is based are determined After grouping the routes into the abovementioned categories, they are filtered through a decision support process that applies the parameter optimization objectives. The optimization model is set so that there is no data in the first group to For the routes in the first group in which the level is set to FED = 0, the model will select the shortest route in length which will represent the optimal evacuation The same optimization process is also set for the second and the third group in which the level is set to FED>0 ≤ 0,5 and FED>0,5 ≤ 0,8 accordingly. The reason why this three groups are separated is to give an advantage in the optimization process to the routes with less CO exposure than on those with shorter lengths. For the routes in the fourth group in which the level is set to FED > 0,8 ≤ 1, the model will select the route with the minimum CO exposure presented in the form of FED. In this group, clinical symptoms of CO exposure predict conditions that can cause difficulties during the evacuation process, and because of this, the optimization is set based on the FED parameter with minimal value. The evacuation routes selected in this group should be treated with caution, and they should be thoroughly analyzed for opportunities to install additional evacuation support systems in cer- For the routes in the fifth group in which the level is set to FED > 1, the model will treat all routes as unsafe for evacuation. If the proposed methodology in this study does not generate data which will fall into the first four groups, then an additional analysis should be performed using the developed ventilation model that shows the movement of smoke and toxic gases through the underground mining facilities. These results could serve to plan the action strategy for the rescue teams Route 3 (rank 1) 0 3282 Route 4 (rank 2) 0 3327 Route 2 (rank 3) 0,24 2082 Route 1 (rank 4) 0,84 2912 Ranked evacuation routes from the optimization process for scenario S-1 with the use of a SCSR. FED Route length [m] using the COHb model from which COHb (%) concentrations in the blood are predicted for the same CO exposure which in turn are related to the clinical symp- 1.Group 1 of evacuation routes with a value of FED = 0 DOI: http://dx.doi.org/10.5772/intechopen.91213 2.Group 2 of evacuation routes with a value of FED>0 ≤ 0,5 3.Group 3 of evacuation routes with a value of FED>0,5 ≤ 0,8 4.Group 4 of evacuation routes with a value of FED>0,8 ≤ 1 5.Group 5 of evacuation routes with a value of FED>1 continue to the next one until the last group is reached. five categories: toms presented in the Table 4. route. tain critical locations. Table 9. 87 Figure 11. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 1. Figure 12. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 2. Figure 13. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 3. Figure 14. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 4. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 The first step in the optimization model is to group the evacuation routes into five categories: 1.Group 1 of evacuation routes with a value of FED = 0 2.Group 2 of evacuation routes with a value of FED>0 ≤ 0,5 3.Group 3 of evacuation routes with a value of FED>0,5 ≤ 0,8 4.Group 4 of evacuation routes with a value of FED>0,8 ≤ 1 5.Group 5 of evacuation routes with a value of FED>1 The values of the FED parameter on which the grouping is based are determined using the COHb model from which COHb (%) concentrations in the blood are predicted for the same CO exposure which in turn are related to the clinical symptoms presented in the Table 4. After grouping the routes into the abovementioned categories, they are filtered through a decision support process that applies the parameter optimization objectives. The optimization model is set so that there is no data in the first group to continue to the next one until the last group is reached. For the routes in the first group in which the level is set to FED = 0, the model will select the shortest route in length which will represent the optimal evacuation route. The same optimization process is also set for the second and the third group in which the level is set to FED>0 ≤ 0,5 and FED>0,5 ≤ 0,8 accordingly. The reason why this three groups are separated is to give an advantage in the optimization process to the routes with less CO exposure than on those with shorter lengths. For the routes in the fourth group in which the level is set to FED > 0,8 ≤ 1, the model will select the route with the minimum CO exposure presented in the form of FED. In this group, clinical symptoms of CO exposure predict conditions that can cause difficulties during the evacuation process, and because of this, the optimization is set based on the FED parameter with minimal value. The evacuation routes selected in this group should be treated with caution, and they should be thoroughly analyzed for opportunities to install additional evacuation support systems in certain critical locations. For the routes in the fifth group in which the level is set to FED > 1, the model will treat all routes as unsafe for evacuation. If the proposed methodology in this study does not generate data which will fall into the first four groups, then an additional analysis should be performed using the developed ventilation model that shows the movement of smoke and toxic gases through the underground mining facilities. These results could serve to plan the action strategy for the rescue teams Table 9. Ranked evacuation routes from the optimization process for scenario S-1 with the use of a SCSR. Figure 11. Fire Safety and Management Awareness Figure 12. Figure 13. Figure 14. 86 Results from the FED and COHb models, for inhalation of CO during evacuation along the route 1. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 2. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 3. Results from the FED and COHb models, for inhalation of CO during evacuation along the route 4. or for a suggestion of additional systems that could help in the evacuation process for those affected by the fire scenario. Table 11 Shows every optimal evacuation route for group 1 based on the fire scenarios generated by the Monte Carlo simulation model. As previously mentioned the simulation process in the VentFIRE™ module is done from the same fire loca- Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios A methodology for determining optimal evacuation routes in case of underground mine fire has been developed based on the results from simulated fire scenarios. The presented methodology can be consistent with the actual situation of the mine because the development of the fire scenarios is based on the risk analysis generated from the current production plans, and the simulation of the developed To address the stochastic nature of the fire scenarios, the methodology implements the Monte Carlo simulation technique to emphasize the fact related to the inability to accurately determine the input parameters for the fire modeling process. From the large list of stochastic input parameters that can have a noticeable effect on the fire scenarios itself, the authors decided to elaborate only on the process of obtaining fire load inputs, from which the size of the fire depends and thus the amount of generated toxic gases. The results of the proposed methodology point to the fact that by treating the stochastic input parameters presented in this chapter in the form of a fire load, the generated conditions influenced the process of deter- The Monte Carlo simulation model with the above-defined parameters which follows the normal distribution is implemented on a case study from "SASA"-R.N. Macedonia mine. After the analysis with the proposed methodology, a fire scenario generated by the mechanization Scooptram ST7 is located which represents the total fire load. The stochastic model is set to generate 20 variations from the fire load that are treated as separate scenarios in the process of determining the optimal evacua- The process of modeling and simulation of the generated fire scenarios is done with the VentFIRE™ module which uses the ventilation network to calculate the movement of the smoke and toxic gases from which the evacuation process The fire parameters obtained from the simulated scenarios are used to calculate The proposed methodology as the main factors influencing the evacuation process treats the inhalation of CO through the evacuation route presented in the form of FED and COHb, factors in the form of tunnel slope, and smoke visibility that In the analysis presented in this chapter, differences in optimal routes for evac- uation were located only in the conditions of SCSR malfunction. The results presented in Table 11 highlight the importance of this additional analysis that is possible only by creating multiple variants of one fire scenario which is actually the underlying purpose of the proposed methodology. In the conditions of using the SCSR, the proposed methodology has determined and confirmed route 3 as optimal for evacuation in all variants of the generated fire scenarios. The results obtained from the conditions of SCSR malfunction located the changes in the optimal evacuation between routes 2 and 4 depending on the variable conditions that determined all the fire scenarios. This approach of analyzing fire scenarios offers certainty in the process of confirming the optimal route as well as locating possibilities for its the optimal evacuation routes for each of the generated scenarios. affect the speed of evacuation and also the SCSR. change depending on the variable fire conditions. scenarios are performed on the ventilation network from the mine. tion for each of the generated scenarios. DOI: http://dx.doi.org/10.5772/intechopen.91213 6. Conclusion and future aspects mining the optimal evacuation routes. tion routes. depends. 89 Table 9 shows the results from the optimization methodology for scenario S-1 in which the routes are sorted by their ranking, taking into account the use of a SCSR. Considering the use of a SCSR, the optimal evacuation route for scenario S-1 is route 3 which has the best rating according to the present methodology. Table 10 shows the results from the optimization methodology taking into account the possibility of malfunction of the SCSR for scenario S-1. The optimal evacuation route for scenario S-1 in which we assumed the malfunction of the SCSR is route 4 which according to the present methodology has the best rating. #### Table 10. Ranked evacuation routes from the optimization process for scenario S-1 without the use of SCSR. #### Table 11. Optimal evacuation route for every fire scenario generated by the Monte Carlo simulation model. #### Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 Table 11 Shows every optimal evacuation route for group 1 based on the fire scenarios generated by the Monte Carlo simulation model. As previously mentioned the simulation process in the VentFIRE™ module is done from the same fire location for each of the generated scenarios. #### 6. Conclusion and future aspects or for a suggestion of additional systems that could help in the evacuation process Table 10 shows the results from the optimization methodology taking into The optimal evacuation route for scenario S-1 in which we assumed the malfunction of the SCSR is route 4 which according to the present methodology has the Optimal route with SCSR Optimal route without the use of SCSR FED Route length [m] Scenario S-2 Route 3 FED = 0 Length = 3282 m Route 2 FED = 0,421 Length = 2082 m Scenario S-3 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,115 Length = 3327 m Scenario S-4 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,112 Length = 3327 m Scenario S-5 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,175 Length = 3327 m Scenario S-6 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,168 Length = 3327 m Scenario S-7 Route 3 FED = 0 Length = 3282 m Route 2 FED = 0,439 Length = 2082 m Scenario S-8 Route 3 FED = 0 Length = 3282 m Route 2 FED = 0,432 Length = 2082 m Scenario S-9 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,165 Length = 3327 m Scenario S-10 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,161 Length = 3327 m Scenario S-10 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,159 Length = 3327 m Scenario S-11 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,174 Length = 3327 m Scenario S-12 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,163 Length = 3327 m Scenario S-13 Route 3 FED = 0 Length = 3282 m Route 2 FED = 0,448 Length = 2082 m Scenario S-14 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,170 Length = 3327 m Scenario S-15 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,169 Length = 3327 m Scenario S-16 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,162 Length = 3327 m Scenario S-17 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,156 Length = 3327 m Scenario S-18 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,171 Length = 3327 m Scenario S-19 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,144 Length = 3327 m Scenario S-19 Route 3 FED = 0 Length = 3282 m Route 4 FED = 0,173 Length = 3327 m Optimal evacuation route for every fire scenario generated by the Monte Carlo simulation model. route 3 which has the best rating according to the present methodology. Route 4 (rank 1) 0,18 3327 Route 2 (rank 2) 0,74 2082 Route 3 (rank 3) 0,69 3282 Route 1 (rank 4) 1,5 2912 Ranked evacuation routes from the optimization process for scenario S-1 without the use of SCSR. account the possibility of malfunction of the SCSR for scenario S-1. Table 9 shows the results from the optimization methodology for scenario S-1 in which the routes are sorted by their ranking, taking into account the use of a SCSR. Considering the use of a SCSR, the optimal evacuation route for scenario S-1 is for those affected by the fire scenario. Fire Safety and Management Awareness best rating. Table 10. Table 11. 88 A methodology for determining optimal evacuation routes in case of underground mine fire has been developed based on the results from simulated fire scenarios. The presented methodology can be consistent with the actual situation of the mine because the development of the fire scenarios is based on the risk analysis generated from the current production plans, and the simulation of the developed scenarios are performed on the ventilation network from the mine. To address the stochastic nature of the fire scenarios, the methodology implements the Monte Carlo simulation technique to emphasize the fact related to the inability to accurately determine the input parameters for the fire modeling process. From the large list of stochastic input parameters that can have a noticeable effect on the fire scenarios itself, the authors decided to elaborate only on the process of obtaining fire load inputs, from which the size of the fire depends and thus the amount of generated toxic gases. The results of the proposed methodology point to the fact that by treating the stochastic input parameters presented in this chapter in the form of a fire load, the generated conditions influenced the process of determining the optimal evacuation routes. The Monte Carlo simulation model with the above-defined parameters which follows the normal distribution is implemented on a case study from "SASA"-R.N. Macedonia mine. After the analysis with the proposed methodology, a fire scenario generated by the mechanization Scooptram ST7 is located which represents the total fire load. The stochastic model is set to generate 20 variations from the fire load that are treated as separate scenarios in the process of determining the optimal evacuation routes. The process of modeling and simulation of the generated fire scenarios is done with the VentFIRE™ module which uses the ventilation network to calculate the movement of the smoke and toxic gases from which the evacuation process depends. The fire parameters obtained from the simulated scenarios are used to calculate the optimal evacuation routes for each of the generated scenarios. The proposed methodology as the main factors influencing the evacuation process treats the inhalation of CO through the evacuation route presented in the form of FED and COHb, factors in the form of tunnel slope, and smoke visibility that affect the speed of evacuation and also the SCSR. In the analysis presented in this chapter, differences in optimal routes for evacuation were located only in the conditions of SCSR malfunction. The results presented in Table 11 highlight the importance of this additional analysis that is possible only by creating multiple variants of one fire scenario which is actually the underlying purpose of the proposed methodology. In the conditions of using the SCSR, the proposed methodology has determined and confirmed route 3 as optimal for evacuation in all variants of the generated fire scenarios. The results obtained from the conditions of SCSR malfunction located the changes in the optimal evacuation between routes 2 and 4 depending on the variable conditions that determined all the fire scenarios. This approach of analyzing fire scenarios offers certainty in the process of confirming the optimal route as well as locating possibilities for its change depending on the variable fire conditions. In order to further improve the methodology, we need to expand our research by introducing the other stochastic variables that may have impact on the evacuation process such as the physical status of mineworkers that is related to age, gender, exercise ability, and response ability. References pp. 1-19 pp. 7-54 (09)60261-1 [1] Conti R, Chasko L, Wiehagen W. Fire DOI: http://dx.doi.org/10.5772/intechopen.91213 [8] Adjiski V. Possibilities for simulating Mirakovski D, Mijalkovski S. Fire risk assessment and computer simulation of fire scenario in underground mines. Studies in Engineering and Technology. 2015;2(1):54-60. DOI: 10.11114/set. Serafimovski D. Prototype model for fire safety system in underground mining. American Journal of Mining and Metallurgy. 2017;4(1):62-67. DOI: [11] Li X, Hadjisophocleous G, Sun X. Sensitivity and uncertainty analysis of a fire spread model with correlated inputs. Procedia Engineering. 2018; 211(2018):403-414. DOI: 10.1016/j. [12] Guanquan C, Jinhui W. Study on probability distribution of fire scenarios [13] Kong D, Johansson N, Hees P, Lu S, Lo S. A Monte Carlo analysis of the effect of heat release rate uncertainty on available safe egress time. Journal of Fire Protection Engineering. 2013; 23(1):5-29. DOI: 10.1177/104239151 [14] Lindström T, Lund D. A Method of Quantifying User Uncertainty in FDS by Report 5309. Sweden: Department of Fire Safety Engineering and Systems Safety, Lund University; 2009. pp. 15-36 Using Monte Carlo Analysis. in risk assessment to emergency evacuation. Reliability Engineering and System Safety. 2012;99:24-32. DOI: 10.1016/j.ress.2011.10.014 2452676 the smoke rollback effect in underground mines using CFD software. GeoScience Engineering. 2014;2014(2):8-18. DOI: 10.2478/gse- [9] Adjiski V, Despodov Z, [10] Adjiski V, Despodov Z, 10.12691/ajmm-4-1-6 proeng.2017.12.029 2014-0008 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios v2i1.825 [3] Ji J, Zhang J, Chen J, Wu S. Computer 1674-5264. DOI: 10.1016/S1674-5264 [4] Chen P, Guo S, Wang Y. Human evacuation affected by smoke 2016;3(1):28-34. DOI: 10.1007/ [6] Wu Q, Xu H, Du Y, Zhang X, Zhao Y. Emergency evacuation simulation system and engineering application for water bursting disaster in mine. Journal of China Coal Society. 2017;42(10):2491-2497 2015.10.001 91 [7] Adjiski V, Mirakovski D, Despodov Z, Mijalkovski S. Simulation and optimization of evacuation routes in case of fire in underground mines. Journal of Sustainable Mining. 2015; 14(3):133-143. DOI: 10.1016/j.jsm. s40789-015-0100-3 05.007 movement in mine fires. International Journal of Coal Science & Technology. [5] Wang L, Wang Y, Cao Q, Li X, Li J, Wu X. A framework for human error risk analysis of coal mine emergency evacuation in China. Journal of Loss Prevention in the Process Industries. 2014;30(2014):113-123. ISSN: 0950-4230. DOI: 10.1016/j.jlp.2014. Underground Mines. Pittsburgh, PA: National Institute for Occupational Safety and Health-NIOSH; 2005. Response Preparedness for [2] Hansen R. Design fires in underground mines. In: Studies in Sustainable Technology 2010:02. Västerås: Mälardalen University; 2010. simulation of evacuation in underground coal mines. Mining Science and Technology (China). 2010;20(5):677-681. ISSN: This research provides a convenient methodology for improving the accuracy of determining the optimal evacuation routes which significantly can increase the safety in underground mines. #### Acknowledgements This work was financially supported by the Faculty of Natural and Technical Sciences—Mining Engineering, "Goce Delchev" University, Shtip, R.N. Macedonia. #### Author details Vancho Adjiski\* and Zoran Despodov Faculty of Natural and Technical Sciences, Mining Engineering, Goce Delchev University, Shtip, R.N. Macedonia \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213* #### References In order to further improve the methodology, we need to expand our research by introducing the other stochastic variables that may have impact on the evacuation process such as the physical status of mineworkers that is related to age, This research provides a convenient methodology for improving the accuracy of determining the optimal evacuation routes which significantly can increase the This work was financially supported by the Faculty of Natural and Technical Sciences—Mining Engineering, "Goce Delchev" University, Shtip, R.N. Macedonia. Faculty of Natural and Technical Sciences, Mining Engineering, Goce Delchev © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, \Address all correspondence to: [email protected] gender, exercise ability, and response ability. safety in underground mines. Fire Safety and Management Awareness* Acknowledgements Author details 90 Vancho Adjiski\* and Zoran Despodov provided the original work is properly cited. University, Shtip, R.N. Macedonia [1] Conti R, Chasko L, Wiehagen W. Fire Response Preparedness for Underground Mines. Pittsburgh, PA: National Institute for Occupational Safety and Health-NIOSH; 2005. pp. 1-19 [2] Hansen R. Design fires in underground mines. In: Studies in Sustainable Technology 2010:02. Västerås: Mälardalen University; 2010. pp. 7-54 [3] Ji J, Zhang J, Chen J, Wu S. Computer simulation of evacuation in underground coal mines. Mining Science and Technology (China). 2010;20(5):677-681. ISSN: 1674-5264. DOI: 10.1016/S1674-5264 (09)60261-1 [4] Chen P, Guo S, Wang Y. Human evacuation affected by smoke movement in mine fires. International Journal of Coal Science & Technology. 2016;3(1):28-34. DOI: 10.1007/ s40789-015-0100-3 [5] Wang L, Wang Y, Cao Q, Li X, Li J, Wu X. A framework for human error risk analysis of coal mine emergency evacuation in China. Journal of Loss Prevention in the Process Industries. 2014;30(2014):113-123. ISSN: 0950-4230. DOI: 10.1016/j.jlp.2014. 05.007 [6] Wu Q, Xu H, Du Y, Zhang X, Zhao Y. Emergency evacuation simulation system and engineering application for water bursting disaster in mine. Journal of China Coal Society. 2017;42(10):2491-2497 [7] Adjiski V, Mirakovski D, Despodov Z, Mijalkovski S. Simulation and optimization of evacuation routes in case of fire in underground mines. Journal of Sustainable Mining. 2015; 14(3):133-143. DOI: 10.1016/j.jsm. 2015.10.001 [8] Adjiski V. Possibilities for simulating the smoke rollback effect in underground mines using CFD software. GeoScience Engineering. 2014;2014(2):8-18. DOI: 10.2478/gse-2014-0008 [9] Adjiski V, Despodov Z, Mirakovski D, Mijalkovski S. Fire risk assessment and computer simulation of fire scenario in underground mines. Studies in Engineering and Technology. 2015;2(1):54-60. DOI: 10.11114/set. v2i1.825 [10] Adjiski V, Despodov Z, Serafimovski D. Prototype model for fire safety system in underground mining. American Journal of Mining and Metallurgy. 2017;4(1):62-67. DOI: 10.12691/ajmm-4-1-6 [11] Li X, Hadjisophocleous G, Sun X. Sensitivity and uncertainty analysis of a fire spread model with correlated inputs. Procedia Engineering. 2018; 211(2018):403-414. DOI: 10.1016/j. proeng.2017.12.029 [12] Guanquan C, Jinhui W. Study on probability distribution of fire scenarios in risk assessment to emergency evacuation. Reliability Engineering and System Safety. 2012;99:24-32. DOI: 10.1016/j.ress.2011.10.014 [13] Kong D, Johansson N, Hees P, Lu S, Lo S. A Monte Carlo analysis of the effect of heat release rate uncertainty on available safe egress time. Journal of Fire Protection Engineering. 2013; 23(1):5-29. DOI: 10.1177/104239151 2452676 [14] Lindström T, Lund D. A Method of Quantifying User Uncertainty in FDS by Using Monte Carlo Analysis. Report 5309. Sweden: Department of Fire Safety Engineering and Systems Safety, Lund University; 2009. pp. 15-36 [15] Salem AM. Use of Monte Carlo simulation to assess uncertainties in fire consequence calculation. Ocean Engineering. 2016;117:411-430. DOI: 10.1016/j.oceaneng.2016.03.050 [16] Adjiski V, Zubicek V, Despodov Z. Monte Carlo simulation of uncertain parameters to evaluate the evacuation process in an underground mine fire emergency. The Southern African Institute of Mining and Metallurgy. 2019;119(11):907-917. DOI: 10.17159/ 2411-9717/701/2019 [17] Gillies S, Wu HW. Case studies from simulating mine fires in coal mines and their effects on mine ventilation systems. In: Aziz N, editor. Coal 2004: Coal Operators' Conference, University of Wollongong & the Australasian Institute of Mining and Metallurgy. 2004. pp. 111-125 [18] Ventsim Visual™ User Guide. Ventsim Software by Chasm Consulting. Capalaba, QLD, Australia; 2014 [19] Adjiski V, Mirakovski D, Despodov Z, Mijalkovski S. CFD simulation of the brattice barrier method for approaching underground mine fires. Mining. Science. 2016;23: 161-172. DOI: 10.5277/ msc162313 [20] Hansen R, Ingason H. Heat release rate measurements of burning mining vehicles in an underground mine. Fire Safety Journal. 2013;61:12-25. DOI: 10.1016/j.firesaf.2013.08.009 [21] Roh JS, Ryou HS, Kim DH. Critical velocity and burning rate in pool fire during longitudinal ventilation. Tunneling Underground Space Technology. 2007;22(3):262-271 [22] Hansen R. Literature survey-fire and smoke spread in underground mines. In: MdH SiST 2009:2. Västerås: Mälardalens Högskola; 2009. pp. 7-67 [23] Purser DA. Modelling toxic and physical hazard in fire. Fire Safety Science. 1989;2:391-400. DOI: 10.3801/ IAFSS.FSS.2-391 [30] Fridolf K, Nilsson D, Frantzich H, Ronchi E, Arias S. Walking speed in smoke: Representation in life safety verifications. In: 12th International Performance-Based Codes and Fire Safety Design Methods Conference, DOI: http://dx.doi.org/10.5772/intechopen.91213 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios Oahu, Hawaii. 2018. pp. 1-6 687969 9458785 pp. 537-548 93 [31] Guangwei Y, Dandan F. Escaperoute planning of underground coal mine based on improved ant algorithm. Mathematical Problems in Engineering. 2013;2013:32-46. DOI: 10.1155/2013/ [32] Ruixin Z, Rongshan N, Hongze Z, Yanqiang F. Experimental study on the escape velocity of miners during mine fire periods. Mathematical Problems in Engineering. 2018;2018:1-12. DOI: 10.1155/2018/9458785. Article ID: [33] Fridolf K, Frantzich H, Ronchi E, Nilsson D. The relationship between obstructed and unobstructed walking speed: Results from an evacuation experiment in a smoke filled tunnel. In: 6th International Symposium on Human Behavior in Fire. Cambridge. 2015. [34] Fridolf K, Ronchi E, Nilsson D, Frantzich H. Movement speed and exit choice in smokefilled rail tunnels. Fire Safety Journal. 2013;59:8-21. DOI: 10.1016/j.firesaf.2013.03.007 [35] McAteer D. The Sago Mine Disaster. Buckhannon, West Virginia; 2016. p. 110. Available from: www.wvgov.org [24] Purser DA. Toxicity assessment of combustion products. In: SFPE Handbook of Fire Protection Engineering. 3rd ed. Quincy, MA: National Fire Protection Association (NFPA); 2002. pp. 2-83 [25] Dirks KN, Sturman A, Johns MD. Using health impacts to assess atmospheric carbon monoxide models. Meteorological Applications. 2006; 13(1):83-87. DOI: 10.1017/ S1350482705002057 [26] Chaloulakou A, Fili N, Spyrelis N. Occupational exposure to CO concentrations in enclosed garages: Estimation of blood COHb levels. In: Environmental Pollution, Proceedings of the 5th International Conference, Thessaloniki, Greece. 2000. pp. 934-940 [27] Coburn RF, Forster RE, Kane PB. Considerations of the physiological variables that determine the blood carboxyhæmoglobin concentrations in man. The Journal of Clinical Investigation. 1965;44:1899-1910. DOI: 10.1172/JCI105296 [28] Adjiski V, Despodov Z, Serafimovski D. System for prediction of carboxyhemoglobin levels as an indicator for on-time installation of selfcontained self-rescuers in case of fire in underground mines. GeoScience Engineering. 2019;65(4):23-37. ISSN: 1802-5420. DOI: 10.35180/gse-2019-0021 [29] Ronchi E, Gwynne SMV, Purser DA. The impact of default settings on evacuation model results: A study of visibility conditions vs occupant walking speeds. In: Advanced Research Workshop - Evacuation and Human Behaviour in Emergency Situations-Santander, Spain. 2011. pp. 2-15 Methodology for Optimal Fire Evacuations in Underground Mines Based on Simulated Scenarios DOI: http://dx.doi.org/10.5772/intechopen.91213 [30] Fridolf K, Nilsson D, Frantzich H, Ronchi E, Arias S. Walking speed in smoke: Representation in life safety verifications. In: 12th International Performance-Based Codes and Fire Safety Design Methods Conference, Oahu, Hawaii. 2018. pp. 1-6 [15] Salem AM. Use of Monte Carlo simulation to assess uncertainties in fire Fire Safety and Management Awareness [23] Purser DA. Modelling toxic and physical hazard in fire. Fire Safety Science. 1989;2:391-400. DOI: 10.3801/ [24] Purser DA. Toxicity assessment of [25] Dirks KN, Sturman A, Johns MD. Using health impacts to assess atmospheric carbon monoxide models. Meteorological Applications. 2006; [26] Chaloulakou A, Fili N, Spyrelis N. [27] Coburn RF, Forster RE, Kane PB. Considerations of the physiological variables that determine the blood carboxyhæmoglobin concentrations in Investigation. 1965;44:1899-1910. DOI: Serafimovski D. System for prediction of carboxyhemoglobin levels as an indicator for on-time installation of selfcontained self-rescuers in case of fire in underground mines. GeoScience Engineering. 2019;65(4):23-37. ISSN: 1802-5420. DOI: 10.35180/gse- [29] Ronchi E, Gwynne SMV, Purser DA. The impact of default settings on evacuation model results: A study of visibility conditions vs occupant walking speeds. In: Advanced Research Workshop - Evacuation and Human Behaviour in Emergency Situations-Santander, Spain. 2011. pp. 2-15 combustion products. In: SFPE Handbook of Fire Protection Engineering. 3rd ed. Quincy, MA: National Fire Protection Association (NFPA); 2002. pp. 2-83 13(1):83-87. DOI: 10.1017/ S1350482705002057 Occupational exposure to CO concentrations in enclosed garages: Estimation of blood COHb levels. In: Environmental Pollution, Proceedings of the 5th International Conference, Thessaloniki, Greece. 2000. pp. 934-940 man. The Journal of Clinical [28] Adjiski V, Despodov Z, 10.1172/JCI105296 2019-0021 IAFSS.FSS.2-391 [16] Adjiski V, Zubicek V, Despodov Z. Monte Carlo simulation of uncertain parameters to evaluate the evacuation process in an underground mine fire emergency. The Southern African Institute of Mining and Metallurgy. 2019;119(11):907-917. DOI: 10.17159/ [17] Gillies S, Wu HW. Case studies from simulating mine fires in coal mines and their effects on mine ventilation systems. In: Aziz N, editor. Coal 2004: Coal Operators' Conference, University of Wollongong & the Australasian Institute of Mining and Metallurgy. [18] Ventsim Visual™ User Guide. Ventsim Software by Chasm [19] Adjiski V, Mirakovski D, Despodov Z, Mijalkovski S. CFD simulation of the brattice barrier method for approaching underground mine fires. Mining. Science. 2016;23: 161-172. DOI: 10.5277/ msc162313 Consulting. Capalaba, QLD, Australia; [20] Hansen R, Ingason H. Heat release rate measurements of burning mining vehicles in an underground mine. Fire Safety Journal. 2013;61:12-25. DOI: 10.1016/j.firesaf.2013.08.009 [21] Roh JS, Ryou HS, Kim DH. Critical velocity and burning rate in pool fire during longitudinal ventilation. Tunneling Underground Space Technology. 2007;22(3):262-271 [22] Hansen R. Literature survey-fire and smoke spread in underground mines. In: MdH SiST 2009:2. Västerås: Mälardalens Högskola; 2009. pp. 7-67 consequence calculation. Ocean Engineering. 2016;117:411-430. DOI: 10.1016/j.oceaneng.2016.03.050 2411-9717/701/2019 2004. pp. 111-125 2014 92 [31] Guangwei Y, Dandan F. Escaperoute planning of underground coal mine based on improved ant algorithm. Mathematical Problems in Engineering. 2013;2013:32-46. DOI: 10.1155/2013/ 687969 [32] Ruixin Z, Rongshan N, Hongze Z, Yanqiang F. Experimental study on the escape velocity of miners during mine fire periods. Mathematical Problems in Engineering. 2018;2018:1-12. DOI: 10.1155/2018/9458785. Article ID: 9458785 [33] Fridolf K, Frantzich H, Ronchi E, Nilsson D. The relationship between obstructed and unobstructed walking speed: Results from an evacuation experiment in a smoke filled tunnel. In: 6th International Symposium on Human Behavior in Fire. Cambridge. 2015. pp. 537-548 [34] Fridolf K, Ronchi E, Nilsson D, Frantzich H. Movement speed and exit choice in smokefilled rail tunnels. Fire Safety Journal. 2013;59:8-21. DOI: 10.1016/j.firesaf.2013.03.007 [35] McAteer D. The Sago Mine Disaster. Buckhannon, West Virginia; 2016. p. 110. Available from: www.wvgov.org 95 Section 4 Safety Protocols with Case Studies Section 4	doab	2025-04-07T03:56:59.078056	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 7 }
006df1b2-f518-4d6b-8d96-484202f13086.8	Safety Protocols with Case Studies 97 Chapter 6 Abstract Agnes Iringová Slovak legislation. 1. Introduction A Case Study on the Fire Safety in This chapter deals with the issue of fire safety in historic buildings that undergo functional change, restoration, replacement of construction, facade or installation renovation. It analyzes the current technical state in relation to microclimate and fire safety in historic buildings in Slovakia. It pays attention to the legislative framework for building conservation in the Slovak Republic considering its impact on the reconstruction and restoration of historic buildings. It assesses approaches and methods for fire safety solutions in historic buildings depending on the extent of their modification—intervention in the layout, function and construction. It presents solution procedures and knowledge in terms of application of fire safety requirements in historic buildings using model examples in accordance with the Keywords: fire safety, restoration, historic buildings, legislation, model examples The successful restoration or renovation of a historic building depends on the integration of new operational requirements into the existing premises without the necessity of changing its original structure, layout and appearance. It is important for the building conservation to preserve the building's originality after its renovation and provide better microclimate and safety standard. The extent of the construction changes is connected with the extent of changes to fire safety solution. Restoration of a historic building can be defined as a set of layout and construction modifications implemented into the building structures in such a way that the building's original height and ground plan can be preserved. The construction interventions modify the building's technical parameters such as layout, load-bearing capacity, thermal and acoustic protection and fire safety. The building proceeding in the Slovak Republic related to the above-mentioned changes follows Act No. Restoration of a historic building can be as follows: (a) an exact restoration, Nowadays, most original historic buildings are not suitable for the occupation; they do not meet either hygiene or static, thermal and fire protection requirements. As for the restoration of a historic building, it is important to pay attention to the based on the detailed documentation of the building's original condition; (b) analogous restoration, based on the verifiable similarity or sameness with a better preserved building; and (c) hypothetical restoration, based on a substantiated, scientifically formulated hypothesis (assumption), giving the base for 50/1976 Coll. on town planning and building code. rebuilding a destroyed or disappeared building or its part. Historic Buildings in Slovakia #### Chapter 6 ## A Case Study on the Fire Safety in Historic Buildings in Slovakia Agnes Iringová #### Abstract This chapter deals with the issue of fire safety in historic buildings that undergo functional change, restoration, replacement of construction, facade or installation renovation. It analyzes the current technical state in relation to microclimate and fire safety in historic buildings in Slovakia. It pays attention to the legislative framework for building conservation in the Slovak Republic considering its impact on the reconstruction and restoration of historic buildings. It assesses approaches and methods for fire safety solutions in historic buildings depending on the extent of their modification—intervention in the layout, function and construction. It presents solution procedures and knowledge in terms of application of fire safety requirements in historic buildings using model examples in accordance with the Slovak legislation. Keywords: fire safety, restoration, historic buildings, legislation, model examples #### 1. Introduction The successful restoration or renovation of a historic building depends on the integration of new operational requirements into the existing premises without the necessity of changing its original structure, layout and appearance. It is important for the building conservation to preserve the building's originality after its renovation and provide better microclimate and safety standard. The extent of the construction changes is connected with the extent of changes to fire safety solution. Restoration of a historic building can be defined as a set of layout and construction modifications implemented into the building structures in such a way that the building's original height and ground plan can be preserved. The construction interventions modify the building's technical parameters such as layout, load-bearing capacity, thermal and acoustic protection and fire safety. The building proceeding in the Slovak Republic related to the above-mentioned changes follows Act No. 50/1976 Coll. on town planning and building code. Restoration of a historic building can be as follows: (a) an exact restoration, based on the detailed documentation of the building's original condition; (b) analogous restoration, based on the verifiable similarity or sameness with a better preserved building; and (c) hypothetical restoration, based on a substantiated, scientifically formulated hypothesis (assumption), giving the base for rebuilding a destroyed or disappeared building or its part. Nowadays, most original historic buildings are not suitable for the occupation; they do not meet either hygiene or static, thermal and fire protection requirements. As for the restoration of a historic building, it is important to pay attention to the choice and optimization of building materials and the optimization of the building's functional use in terms of fire safety. In these cases, the fire safety measures should be the result of a compromise among the fire protection, building conservation, building law and quality requirements for the building's new function. Fire safety in historic buildings is applied using passive and active fire protection of spaces and structures. #### 2. Analysis of the current fire safety in historic buildings in Slovakia Based on the data from fire and rescue corps from 2013 to 2017, there are 2450 buildings of historic significance in Slovakia. Nowadays, the fire safety in historic buildings of great national and cultural importance (e.g. castles, cathedrals, mansions, etc.) where many rare museum exhibits are located is provided by electric fire signalization (EFS) equipment, fire extinguishers, internal and external fire cocks and firefighting measures applied in the building's operation. These measures are related to the fire training of employees who stay in the building during the operation. Each employee is trained how to eliminate fire in its initial phase, evacuate persons and exhibits and call the firefighters. As stated in the Act of the Slovak Ministry of Interior No. 199/2009 on fire protection as amended, the building's operator is obliged to work up, keep and maintain the fire documentation according to the current condition and ensure it is respected. Each owner or administrator of a listed building should determine a qualified person who will be responsible for respecting all operational and organizational measures related to fire safety in a building and will keep and update the fire documentation. Trained persons provide and take regular prophylactic fire inspections of firefighting equipment. Fire brigades regularly carry out training exercises in significant listed buildings to check their firefighting skills, means and methods and the accessibility of fire equipment in buildings. Despite the above measures, the real fire protection in most historic buildings in Slovakia is weak, and fire alarm systems are not located in all buildings. As Fire and Rescue Service Report 2018 states, there were 40 fires in such heritage buildings in the last 10 years. The most common cause was negligence, technical failure or deliberateness combined with the fire risk at the time of the building's operation. Analysis of fire safety in listed buildings is primarily focused on firefighting equipment in terms of its location, availability and functionality as well as on staff readiness to use it effectively [1]. The most common deficiencies found during the fire inspections or in analyses of fire causes in such buildings are as follows: 99 Figure 1. present time. A Case Study on the Fire Safety in Historic Buildings in Slovakia • Insufficient maintenance of public spaces in terms of fire spread, location and storage of flammable materials in the immediate vicinity of the building • Improper handling with the heat or ignition source, that is heaters, welding kits and handling with an open fire, where smoking is prohibited, etc. It is not always possible to prevent fire in a building despite the implementation of fire-protective and operational measures, especially in unforeseeable natural disasters. In general, if fire safety measures are kept at all levels of protection, it is supposed that the building does not collide with fire. If fire safety measures are missing, neglected or non-functional at the time of fire, it often causes big artistic Here is the example of fire in the castle of Krásna Hôrka from 2012. The fire progress is shown in Figure 1b. Fire was caused by children who carelessly handled free flame near the castle hill. They threw a burning object into dry grass that ignited. As there was strong wind, fire spread rapidly onto the combustible castle roofs covered by wooden shingles (see Figure 1a). The castle consists of three buildings. The original upper castle dates from the fourteenth century; the middle and lower castles were built later by the original owners. The castle housed a permanent display of period works of art giving basic information on the castle and its original owners. There were original exhibits with a high museum value. The original roof structure consisted of timber trusses covered with wooden shingles and took the castle has steel-bearing ceiling structure with a wooden flap. The castle has stone Fire safety in the castle before fire included passive fire protection, roof space had no accidental fire load, and timber truss members were treated with fire coating The castle of Krásna Hôrka before fire, during fire, after fire and at the present time. (a) Original castle timber roofs before fire in 2012; (b) fire in the castle in 2012; (c) the castle after fire in 2012; (d) the castle at the . The lower and middle castle has vaulted ceilings; the upper • Technical defects in electrical installations or other equipment DOI: http://dx.doi.org/10.5772/intechopen.91241 • Incendiarism, vandalism and architectural losses. area of about 5000 m<sup>2</sup> and brick external walls. Fire Safety and Management Awareness functional use in terms of fire safety. persons and exhibits and call the firefighters. inspections of firefighting equipment. sibility at the time of fire • Missing or non-functional electrical fire alarm and reinforcement or badly designed crossroads • Missing, capacity-insufficient or unmaintained fire hydrants building's operation. choice and optimization of building materials and the optimization of the building's In these cases, the fire safety measures should be the result of a compromise among the fire protection, building conservation, building law and quality requirements for the building's new function. Fire safety in historic buildings is applied 2. Analysis of the current fire safety in historic buildings in Slovakia Based on the data from fire and rescue corps from 2013 to 2017, there are 2450 buildings of historic significance in Slovakia. Nowadays, the fire safety in historic buildings of great national and cultural importance (e.g. castles, cathedrals, mansions, etc.) where many rare museum exhibits are located is provided by electric fire signalization (EFS) equipment, fire extinguishers, internal and external fire cocks and firefighting measures applied in the building's operation. These measures are related to the fire training of employees who stay in the building during the operation. Each employee is trained how to eliminate fire in its initial phase, evacuate As stated in the Act of the Slovak Ministry of Interior No. 199/2009 on fire protection as amended, the building's operator is obliged to work up, keep and maintain the fire documentation according to the current condition and ensure it is respected. Each owner or administrator of a listed building should determine a qualified person who will be responsible for respecting all operational and organizational measures related to fire safety in a building and will keep and update the fire documentation. Trained persons provide and take regular prophylactic fire Fire brigades regularly carry out training exercises in significant listed buildings to check their firefighting skills, means and methods and the accessibility of fire equipment in buildings. Despite the above measures, the real fire protection in most historic buildings in Slovakia is weak, and fire alarm systems are not located in all buildings. As Fire and Rescue Service Report 2018 states, there were 40 fires in such heritage buildings in the last 10 years. The most common cause was negligence, technical failure or deliberateness combined with the fire risk at the time of the Analysis of fire safety in listed buildings is primarily focused on firefighting equipment in terms of its location, availability and functionality as well as on staff readiness to use it effectively [1]. The most common deficiencies found during the fire inspections or in analyses of fire causes in such buildings are as follows: • Non-functional hand fire extinguishers or their bad location in terms of acces- • Access roads badly rideable for the fire brigade due to insufficient road width • Employees inadequately trained for firefighting and missing fire documentation determining evacuation plans for employees, visitors or exhibits using passive and active fire protection of spaces and structures. 98 It is not always possible to prevent fire in a building despite the implementation of fire-protective and operational measures, especially in unforeseeable natural disasters. In general, if fire safety measures are kept at all levels of protection, it is supposed that the building does not collide with fire. If fire safety measures are missing, neglected or non-functional at the time of fire, it often causes big artistic and architectural losses. Here is the example of fire in the castle of Krásna Hôrka from 2012. The fire progress is shown in Figure 1b. Fire was caused by children who carelessly handled free flame near the castle hill. They threw a burning object into dry grass that ignited. As there was strong wind, fire spread rapidly onto the combustible castle roofs covered by wooden shingles (see Figure 1a). The castle consists of three buildings. The original upper castle dates from the fourteenth century; the middle and lower castles were built later by the original owners. The castle housed a permanent display of period works of art giving basic information on the castle and its original owners. There were original exhibits with a high museum value. The original roof structure consisted of timber trusses covered with wooden shingles and took the area of about 5000 m<sup>2</sup> . The lower and middle castle has vaulted ceilings; the upper castle has steel-bearing ceiling structure with a wooden flap. The castle has stone and brick external walls. Fire safety in the castle before fire included passive fire protection, roof space had no accidental fire load, and timber truss members were treated with fire coating #### Figure 1. The castle of Krásna Hôrka before fire, during fire, after fire and at the present time. (a) Original castle timber roofs before fire in 2012; (b) fire in the castle in 2012; (c) the castle after fire in 2012; (d) the castle at the present time. in 2000, and active fire protection—electric fire signalization—is installed in the roof space [2]. The Gothic tower contained a water reservoir of about 66 m3 ; the upper castle contained fire-water hose systems and wall hydrants. Powder fire extinguishers were installed in all spaces. Although the castle was protected at the time of fire by both passive and active fire protection, its protection was not sufficient considering the outside source of fire, climatic conditions and burning rate of dried roof timber. The fire lasted for about 3 days in terms of the quantity of timber structures and unfavorable natural conditions—strong wind. The roofs burned down (see Figure 1c). The firefighting was slowed down due to the road that was badly accessible for the fire brigade—there is only one access road leading to the castle. The water source was far from the burning area, and it was not possible to use the water reservoir in the castle. The fire affected mainly the Gothic castle that was restored in 1982. The part of the ceiling fell down; some exhibits such as swords and other historical weapons were destroyed. The interior exhibits in the lower and middle castle and the Francis Museum survived without harm. Overall, 90% of all historic exhibits were saved. The castle building suffered fire damage especially on its construction, and the total damage was estimated at approx. 8.05 million €. Nowadays, the castle's restoration is coming to an end. All roofs, including loadbearing and truss structures, were built as replicas of the original structures, taking into account the forms they had at the time of the last major castle's style alternation after fire in 1817 (1818). The original wooden shingle roof is replaced by burnt ceramic roofing, and the bastions have metal roofing (see Figure 1d). In this case, fire was caused by negligence and climatic conditions. #### 3. Past and contemporary legislative regulations for fire safety solutions in historic buildings in Slovakia #### 3.1 Legislative regulations in the past No legislative standards were applied to the construction of buildings in terms of fire protection in the Middle Ages. The fire protection criteria in buildings with timber load-bearing structures were set out in the regulation issued probably by William I. Conqueror (1028–1087). All fireplaces in buildings were required to be put out at night and in the absence of persons. Furthermore, this regulation was supplemented by the requirement to cover the fireplace to prevent air access to the hot ash [3]. It is known from history that after the fire outbreak in the settlement, the consequences were global and fatal for inhabitants due to the combustible roofs and limited possibilities of firefighting at the time. For this reason, the past legislation focused on the fire protection in buildings due to the high risk of easy and rapid fire spreading from building to building. The oldest legislation valid in our territory for the royal free cities, as well as the towns and villages of higher importance with an authorized municipal office that deserved to be added to the royal free cities, was "Fire Regulations for the Kingdom of Hungary" issued in Presburg in 1788. This regulation was divided into four chapters: 1.How to prevent the occurrence of fire—related to the rules for construction of chimneys and internal fireplaces 101 changes. buildings. A Case Study on the Fire Safety in Historic Buildings in Slovakia 3.How to extinguish fire as quickly as possible—each settlement was obliged to have a public water reservoir, pond, lime trees planted on four sides of neigh- Growth in the manufacturing sector in the late nineteenth century brings the use of technique for firefighting. Fire protection starts to be provided by professional fire brigades. Non-combustible building materials—reinforced concrete, burnt ceramic blocks, etc.—are used for the construction of buildings that have natural protection against fire spreading within the building as well as from one building to another. The timber load-bearing elements of ceilings were protected by plasters and embankments made of non-combustible materials. Wooden shingles, straw and reed on the roofs were replaced by ceramic roofing. The timber trusses were separated from chimneys and treated with fire-resistant coatings to reduce their flammability. There was no fire risk in the roof spaces with trusses; they were 3.2 Contemporary legislative regulations for fire safety solutions in historic The obligatory regulation for fire protection currently valid in Slovakia is Act No. 314/2001 as amended and implementary regulation issued by the Slovak Ministry of the Interior No. 121/2002 Coll. on fire prevention, as amended. The implementary regulation No. 94/2004 as amended specifies requirements for the project solution. The restoration of historic buildings takes into account mainly all society's requirements for the preservation of their original appearance and material solutions considering adequate fire safety. Legislation valid for the restoration of historic buildings in Slovakia is Act No. 49/2002 on the heritage protection as amended, issued by the Slovak National Council and followed by the implementary regulations. Details on the performance of monument research are specified in the implementary regulation No. 253/2010 Coll. issued by the Slovak Ministry of Culture. It determines, based on monumental survey, the conditions for methods and extent that can be used in the remediation of existing historic buildings. Survey conclusions are one of the bases for the design and extent of construction work as well as the choice of materials used in the renovation. The requirements and conditions for the restoration of historic buildings in terms of fire safety are limited due to the specific conditions. The restoration and renovation of buildings in Slovakia follows the criteria specified in Slovak Standard STN 730834 on construction In terms of fire safety, the building's alternation is the only alternation resulting in a higher fire risk, number of persons, replacement of load-bearing structures and installations within the affected spaces. The extent of fire safety measures is determined by the extent of changes in the building's construction or operation [4]. The first category includes alternations without the functional change resulting in the higher fire risk. There are only minor repairs to the original structures done without changing their reaction to fire and modernization of installation systems in The second category includes alternations to the functional use of the building's part or the entire building that will change the fire risk, fire resistance requirements for the fire-separating structures, number of people and related evacuation plan. Such alternations to the buildings are related to the fire compartmentation, fire The alternations of buildings can be divided into three categories: 4.How to prevent harmful consequences that may occur after fire DOI: http://dx.doi.org/10.5772/intechopen.91241 boring farm houses, etc. separated from vertical shafts. buildings in Slovakia 2.How to detect fire early if it occurs—signals generated by bells Fire Safety and Management Awareness roof space [2]. reservoir in the castle. damage was estimated at approx. 8.05 million €. fire was caused by negligence and climatic conditions. in historic buildings in Slovakia 3.1 Legislative regulations in the past fire spreading from building to building. chimneys and internal fireplaces in 2000, and active fire protection—electric fire signalization—is installed in the contained fire-water hose systems and wall hydrants. Powder fire extinguishers were installed in all spaces. Although the castle was protected at the time of fire by both passive and active fire protection, its protection was not sufficient considering the outside source of fire, climatic conditions and burning rate of dried roof timber. The fire lasted for about 3 days in terms of the quantity of timber structures and unfavorable natural conditions—strong wind. The roofs burned down (see Figure 1c). The firefighting was slowed down due to the road that was badly accessible for the fire brigade—there is only one access road leading to the castle. The water source was far from the burning area, and it was not possible to use the water The fire affected mainly the Gothic castle that was restored in 1982. The part of the ceiling fell down; some exhibits such as swords and other historical weapons were destroyed. The interior exhibits in the lower and middle castle and the Francis Museum survived without harm. Overall, 90% of all historic exhibits were saved. The castle building suffered fire damage especially on its construction, and the total Nowadays, the castle's restoration is coming to an end. All roofs, including loadbearing and truss structures, were built as replicas of the original structures, taking into account the forms they had at the time of the last major castle's style alternation after fire in 1817 (1818). The original wooden shingle roof is replaced by burnt ceramic roofing, and the bastions have metal roofing (see Figure 1d). In this case, 3. Past and contemporary legislative regulations for fire safety solutions No legislative standards were applied to the construction of buildings in terms of fire protection in the Middle Ages. The fire protection criteria in buildings with timber load-bearing structures were set out in the regulation issued probably by William I. Conqueror (1028–1087). All fireplaces in buildings were required to be put out at night and in the absence of persons. Furthermore, this regulation was supplemented by the requirement to cover the fireplace to prevent air access to the hot ash [3]. It is known from history that after the fire outbreak in the settlement, the consequences were global and fatal for inhabitants due to the combustible roofs and limited possibilities of firefighting at the time. For this reason, the past legislation focused on the fire protection in buildings due to the high risk of easy and rapid The oldest legislation valid in our territory for the royal free cities, as well as the towns and villages of higher importance with an authorized municipal office that deserved to be added to the royal free cities, was "Fire Regulations for the Kingdom of Hungary" issued in Presburg in 1788. This regulation was divided into four 1.How to prevent the occurrence of fire—related to the rules for construction of 2.How to detect fire early if it occurs—signals generated by bells ; the upper castle The Gothic tower contained a water reservoir of about 66 m3 100 chapters: Growth in the manufacturing sector in the late nineteenth century brings the use of technique for firefighting. Fire protection starts to be provided by professional fire brigades. Non-combustible building materials—reinforced concrete, burnt ceramic blocks, etc.—are used for the construction of buildings that have natural protection against fire spreading within the building as well as from one building to another. The timber load-bearing elements of ceilings were protected by plasters and embankments made of non-combustible materials. Wooden shingles, straw and reed on the roofs were replaced by ceramic roofing. The timber trusses were separated from chimneys and treated with fire-resistant coatings to reduce their flammability. There was no fire risk in the roof spaces with trusses; they were separated from vertical shafts. #### 3.2 Contemporary legislative regulations for fire safety solutions in historic buildings in Slovakia The obligatory regulation for fire protection currently valid in Slovakia is Act No. 314/2001 as amended and implementary regulation issued by the Slovak Ministry of the Interior No. 121/2002 Coll. on fire prevention, as amended. The implementary regulation No. 94/2004 as amended specifies requirements for the project solution. The restoration of historic buildings takes into account mainly all society's requirements for the preservation of their original appearance and material solutions considering adequate fire safety. Legislation valid for the restoration of historic buildings in Slovakia is Act No. 49/2002 on the heritage protection as amended, issued by the Slovak National Council and followed by the implementary regulations. Details on the performance of monument research are specified in the implementary regulation No. 253/2010 Coll. issued by the Slovak Ministry of Culture. It determines, based on monumental survey, the conditions for methods and extent that can be used in the remediation of existing historic buildings. Survey conclusions are one of the bases for the design and extent of construction work as well as the choice of materials used in the renovation. The requirements and conditions for the restoration of historic buildings in terms of fire safety are limited due to the specific conditions. The restoration and renovation of buildings in Slovakia follows the criteria specified in Slovak Standard STN 730834 on construction changes. In terms of fire safety, the building's alternation is the only alternation resulting in a higher fire risk, number of persons, replacement of load-bearing structures and installations within the affected spaces. The extent of fire safety measures is determined by the extent of changes in the building's construction or operation [4]. The alternations of buildings can be divided into three categories: The first category includes alternations without the functional change resulting in the higher fire risk. There are only minor repairs to the original structures done without changing their reaction to fire and modernization of installation systems in buildings. The second category includes alternations to the functional use of the building's part or the entire building that will change the fire risk, fire resistance requirements for the fire-separating structures, number of people and related evacuation plan. Such alternations to the buildings are related to the fire compartmentation, fire protection, changes in ventilation system, fire separation of evacuation routes and requirements for the installation of firefighting equipment. The third category includes restorations of buildings, changing the use, useful area and fire height. This is related to the buildings where more than 50% of the total floor area in the fire section changed is found in the building's extension or superstructure [1]. In such cases, the fire safety measures are required to be done completely as in the new buildings, and their assessment is also in accordance with the legislation applicable to the new buildings. The building's functional change often brings the exchange of a building's owner or manager whose criteria for the heat-moisture regime in the indoor environment are higher. As a result of this change, there is a requirement to increase building's thermal protection if the building conservationists give the permit. Thermal protection in historic buildings is done at least to eliminate microclimate deficiencies, optimally considering the building's energy efficiency and sustainability in terms of its environmental impact [5]. If listed buildings are restored, the fire safety solution must contain an expert opinion analyzing the specific building's conditions and determining requirements for its fire safety depending on the boundary conditions such as functional use, design, layout in the vertical direction, occupancy, number and quality of emergency routes, availability of access roads and firefighting water. The fire safety solution should take into account at least the following requirements: the operations with the high fire load and fire factor higher than 1.1, except theaters, exhibition halls, museums and areas for visitors, cannot be situated in the listed buildings whose original function of spaces is modified. The fire alarm systems are required to be installed in the unique historic spaces, e.g. spaces containing murals, unique historic collections, unique structures or elements made of flammable materials. The fire safety reassessment is required to be done if alternations to historic buildings result in their restoration or renewal. #### 4. Theoretical analysis of physical, design and layout determinants affecting the restoration of historic buildings in terms of fire safety Historic buildings were usually constructed using a combination of combustible and non-combustible materials. The most used building material was wood—in roof structures, ceilings and stairs. It was used in the past as a single building material to construct buildings of folk architecture in Slovakia. Historic buildings usually contain composite construction systems. The cellars and basements had stone or masonry walls, and ceilings had ceramic vaults. The above-ground floors had peripheral walls that were built using a combination of non-combustible masonry made of burnt and non-burnt bricks or stone and combustible wood-beamed ceilings. The ceilings were either visible or covered with plaster usually applied to the reed mats. Roof load-bearing structures contained roof trusses with wooden purlins statically independent on the last floor ceiling. Depending on the building's ground plan dimensions, the purlin or collar systems were mostly used for small spans in folk architecture; a combination of standing saddles and hanging trusses or strut frames was used for larger spans, e.g. mansions, castles or churches. The roof space was usually naturally ventilated and had no functional use. The attic was accessible via wooden or stone single or spiral stairs due to the repairs and maintenance. The wooden ceilings and trusses as well as the dimensions of their members were based primarily on the spans they covered and empirical and technical possibilities of the builders at the time of construction. Due to the 103 fire resistance. A Case Study on the Fire Safety in Historic Buildings in Slovakia technical possibilities of the joints affecting the load-bearing capacity of the purlin system, the wooden members were dimensioned with a significant static reserve. The wooden members were usually joined by mortising or lapping, and their fire The fire safety degree is determined on the basis of fire load density with dependence on the ventilation parameter, fire risk, building's fire height and combustibility of used building elements according to Table 8 STN 730802/10. The degree of fire safety in building structures (DFSB) value is the basis for determining fire safety requirements of load-bearing and fire-separating structures given in Table 12 STN 730802/10. These requirements are compared to the current fire resistance of The fire resistance of the original structures can be taken from the table in STN Fire resistance is the ability of building structures to withstand the effect of fire. It is defined by the time during which the structures can be exposed to fire without damaging their function. The fire structures can be divided into loadbearing and non-load-bearing, in terms of their function, and fire-separating or interior load-bearing, in terms of their location in a fire compartment. If the fire-separating structure is load-bearing and located at the frontier between fire compartments, it must meet the criteria of load-bearing capacity (R), integrity (E) and thermal insulation (I) at the time of fire. If the load-bearing structure within the fire compartment is a post, it must meet the R criterion at the required time. The stability of fire structures along the building's height must not depend on the stability of structures with lower fire resistance on lower floors. The fire resistance of fire-separating structures is determined by a test or calculation. The design and assessment of fire resistance of building structures follow a set of standards— Eurocodes EN 1991-1-2, EN 1992-1-2, EN 1993-1-2, EN 1994-1-2, EN 1995-1-2, EN The fire resistance of building structures is calculated using the design procedure in terms of the requirement for the result accuracy and specific boundary conditions of a fire compartment. First, thermal analysis of a fire compartment is done, then the heat transfer into the structure and temperature development within the structure is determined, and finally the fire-separating structure is analyzed. Detailed analyses of the temperature in a fire compartment are determined by dynamic simulations and end-element methods. Simpler procedures are used to determine the temperature in a fire compartment by parametric temperature curves or nominal temperature curves. The resulting fire resistance determined according to the nominal standard curves is the standard fire resistance (Figure 2). The heat transfer within the structure for detailed solutions is determined by the end-element method; for less detailed solutions, it is determined by incremental or direct methods. Direct methods used for heat transfer are conservative and valid only to a limited extent and can be used to assess only particular elements of a fire-separating structure. The calculation is based on room temperature [7–9]. Fire resistance verification of a fire-separating structure can be done by the three following views: Time—clearly expresses the reliability reserves of the structural element: where tfi,d is the design time of fire resistance and tfi,requ is the required time of tfi,d≥ tfi,requ (1) 730821 or calculated according to Eurocodes depending on their static stress. 4.1 Fire resistance of load-bearing and fire-separating structures DOI: http://dx.doi.org/10.5772/intechopen.91241 resistance was achieved by partial walling [6]. the existing structures. 1996-1-2 and EN 1999-1-2. A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 Fire Safety and Management Awareness its environmental impact [5]. protection, changes in ventilation system, fire separation of evacuation routes and The third category includes restorations of buildings, changing the use, useful area and fire height. This is related to the buildings where more than 50% of the total floor area in the fire section changed is found in the building's extension or superstructure [1]. In such cases, the fire safety measures are required to be done completely as in the new buildings, and their assessment is also in accordance with The building's functional change often brings the exchange of a building's owner or manager whose criteria for the heat-moisture regime in the indoor environment are higher. As a result of this change, there is a requirement to increase building's thermal protection if the building conservationists give the permit. Thermal protection in historic buildings is done at least to eliminate microclimate deficiencies, optimally considering the building's energy efficiency and sustainability in terms of If listed buildings are restored, the fire safety solution must contain an expert opinion analyzing the specific building's conditions and determining requirements for its fire safety depending on the boundary conditions such as functional use, design, layout in the vertical direction, occupancy, number and quality of emergency routes, availability of access roads and firefighting water. The fire safety solution should take into account at least the following requirements: the operations with the high fire load and fire factor higher than 1.1, except theaters, exhibition halls, museums and areas for visitors, cannot be situated in the listed buildings The fire alarm systems are required to be installed in the unique historic spaces, Historic buildings were usually constructed using a combination of combustible and non-combustible materials. The most used building material was wood—in roof structures, ceilings and stairs. It was used in the past as a single building material to construct buildings of folk architecture in Slovakia. Historic buildings usually contain composite construction systems. The cellars and basements had stone or masonry walls, and ceilings had ceramic vaults. The above-ground floors had peripheral walls that were built using a combination of non-combustible masonry made of burnt and non-burnt bricks or stone and combustible wood-beamed ceilings. The ceilings were either visible or covered with plaster usually applied to the reed mats. Roof load-bearing structures contained roof trusses with wooden purlins statically independent on the last floor ceiling. Depending on the building's ground plan dimensions, the purlin or collar systems were mostly used for small spans in folk architecture; a combination of standing saddles and hanging trusses or strut The roof space was usually naturally ventilated and had no functional use. The attic was accessible via wooden or stone single or spiral stairs due to the repairs and maintenance. The wooden ceilings and trusses as well as the dimensions of their members were based primarily on the spans they covered and empirical and technical possibilities of the builders at the time of construction. Due to the e.g. spaces containing murals, unique historic collections, unique structures or 4. Theoretical analysis of physical, design and layout determinants affecting the restoration of historic buildings in terms of fire safety frames was used for larger spans, e.g. mansions, castles or churches. The fire safety reassessment is required to be done if alternations to historic requirements for the installation of firefighting equipment. the legislation applicable to the new buildings. whose original function of spaces is modified. buildings result in their restoration or renewal. elements made of flammable materials. 102 technical possibilities of the joints affecting the load-bearing capacity of the purlin system, the wooden members were dimensioned with a significant static reserve. The wooden members were usually joined by mortising or lapping, and their fire resistance was achieved by partial walling [6]. The fire safety degree is determined on the basis of fire load density with dependence on the ventilation parameter, fire risk, building's fire height and combustibility of used building elements according to Table 8 STN 730802/10. The degree of fire safety in building structures (DFSB) value is the basis for determining fire safety requirements of load-bearing and fire-separating structures given in Table 12 STN 730802/10. These requirements are compared to the current fire resistance of the existing structures. The fire resistance of the original structures can be taken from the table in STN 730821 or calculated according to Eurocodes depending on their static stress. #### 4.1 Fire resistance of load-bearing and fire-separating structures Fire resistance is the ability of building structures to withstand the effect of fire. It is defined by the time during which the structures can be exposed to fire without damaging their function. The fire structures can be divided into loadbearing and non-load-bearing, in terms of their function, and fire-separating or interior load-bearing, in terms of their location in a fire compartment. If the fire-separating structure is load-bearing and located at the frontier between fire compartments, it must meet the criteria of load-bearing capacity (R), integrity (E) and thermal insulation (I) at the time of fire. If the load-bearing structure within the fire compartment is a post, it must meet the R criterion at the required time. The stability of fire structures along the building's height must not depend on the stability of structures with lower fire resistance on lower floors. The fire resistance of fire-separating structures is determined by a test or calculation. The design and assessment of fire resistance of building structures follow a set of standards— Eurocodes EN 1991-1-2, EN 1992-1-2, EN 1993-1-2, EN 1994-1-2, EN 1995-1-2, EN 1996-1-2 and EN 1999-1-2. The fire resistance of building structures is calculated using the design procedure in terms of the requirement for the result accuracy and specific boundary conditions of a fire compartment. First, thermal analysis of a fire compartment is done, then the heat transfer into the structure and temperature development within the structure is determined, and finally the fire-separating structure is analyzed. Detailed analyses of the temperature in a fire compartment are determined by dynamic simulations and end-element methods. Simpler procedures are used to determine the temperature in a fire compartment by parametric temperature curves or nominal temperature curves. The resulting fire resistance determined according to the nominal standard curves is the standard fire resistance (Figure 2). The heat transfer within the structure for detailed solutions is determined by the end-element method; for less detailed solutions, it is determined by incremental or direct methods. Direct methods used for heat transfer are conservative and valid only to a limited extent and can be used to assess only particular elements of a fire-separating structure. The calculation is based on room temperature [7–9]. Fire resistance verification of a fire-separating structure can be done by the three following views: Time—clearly expresses the reliability reserves of the structural element: $$\mathbf{t}\_{\text{fi,dz}} \mathbf{t}\_{\text{fi,requ}} \tag{1}$$ where tfi,d is the design time of fire resistance and tfi,requ is the required time of fire resistance. Figure 2. Surface temperature on fire-separating structures without surface fire protection and with fire-protective lining during standard fire [10]. Load-bearing capacity—the easiest in terms of calculation because the method is similar to the assessment at the room temperature: $$\mathbf{R}\_{\text{fi,d,t}} \succeq \mathbf{E}\_{\text{fi,d,t}} \tag{2}$$ Rfi,d,t is the design value of load-bearing capacity of a member in fire during the time t and Efi,d,t is the design value of fire load effects during the time t. Temperature: $$ \Theta\_{\mathbf{d}} \succeq \Theta\_{\mathbf{cr,d}} \tag{3} $$ 105 731701 Table 1. A Case Study on the Fire Safety in Historic Buildings in Slovakia fire safety degree was not changed compared to the original one. The attic spaces used as living rooms serve today only to show the original truss construction. Considering the fire height of 0 m and the combined building's construction, the required fire safety degree is I, that is the same as in the original functional use. The fire resistance requirement for the original load-bearing and fire-separating structures was not changed after the functional change of the restored spaces. The fire resistance requirement for the load-bearing ceiling members and perimeter wall is given according to STN 73082 and is dependent on the average fire load, that is the sum of the accidental and permanent fire load, coefficients of ventilation, flammability factor and use and type of firefighting equipment. In terms of the calculated fire REI 30 (building envelope and roof), and the fire resistance requirement for loadbearing structures of a non-compact ceiling in an assessed fire compartment is R 30. The external wall is made of stone and brick and has a variable thickness of 530–630 mm. The required fire resistance for this model solution is REI 30 min. In accordance with the values given in Table 1A STN 730821, the real fire resis- As the dimensions of the assessed wooden ceiling members and column in a model solution are different from the members given in the standard, their real fire resistance is calculated according to EN 1996-1-2: 2004 Eurocode 6: Design of masonry structures, Section 1.2 general rules—fire resistance design of masonry In specific cases, the fire resistance of fire-separating structures can be determined by a calculation according to EN 1996-1-2: 2004 Eurocode 6: Design of masonry structures, Section 1.2 general rules—fire resistance design of masonry There is a wooden beamed ceiling above the ground plan in the model example. The ceiling beams are supported by a wooden beam. The wooden beam is fastened on the load-bearing peripheral walls and supported by a wooden column The real fire resistance for load-bearing and fire-separating structures was calculated according to the methodology given in STN EN 1991-1-2: 2004; the Type of a structural member Thickness [mm] Fire Wooden beams loaded in bending, unprotected from three sides 140/200 40 resistance REI >180 240 200/200 20 4.3 Fire resistance of the external wall in a model solution tance of the perimeter wall is well above the required value (Table 1). 4.4 Fire resistance of the wooden ceiling in a model solution The ceiling material and structure are visible (see Figure 3). Masonry walls made of solid bricks perforated up to 15% of the volume, built on mortar of 4-CSN 2430 class, loaded and non-loaded Unprotected wooden columns loaded in buckling at λ = 75, see CSN Fire resistance of the fire-separating structures according to STN 730821 [12]. , the fire resistance requirement for the fire-separated structures is DOI: http://dx.doi.org/10.5772/intechopen.91241 load pv = 66 kg/m2 structures. structures. (see Figure 4). with double-sided plaster where θd is the design value of material temperature and θcr,d is the design value of critical material temperature. Simplified assessment of structural elements in terms of their fire resistance is given in tables in STN 730821: 1973, which is currently valid for the assessment of building structures during construction changes. The fire resistance values of building structures are given in particular tables considering building materials and static load of the structures—walls, columns and ceilings [11]. #### 4.2 Fire resistance assessment of existing fire-separating structures in a model solution The following model example shows a fire safety solution used in the restoration of a folk house situated in the village of Vel'ké Leváre. The folk house is dated to the Hutterian culture period. It was restored with the intention of preserving its original layout including the original constructions and elements. The building has a combined structural system—the brick external walls, wooden beam ceiling and collar beam truss. It was necessary to optimize the boundary conditions of the given solution so that the consequences of a functional change regarding the current constructions could be minimal. The museum display showing the original culture was situated in the restored space after the original supporting elements, roof covering and original wall and floor surfaces had been replaced or repaired. The external wall is combined stone with bricks. There are wooden ceilings with visible beams supported by a wooden beam. This beam is embedded into the perimeter walls, and its center is supported by a column. The wooden truss has a two-level collar beam. The building's functional use was changed in terms of fire safety—it became a museum, that is its original residential function was changed into an exhibition one. The fire load increased but only on the first floor. The required value of the #### A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 Fire Safety and Management Awareness similar to the assessment at the room temperature: Temperature: during standard fire [10]. Figure 2. solution two-level collar beam. of critical material temperature. Load-bearing capacity—the easiest in terms of calculation because the method is Surface temperature on fire-separating structures without surface fire protection and with fire-protective lining Rfi,d,t is the design value of load-bearing capacity of a member in fire during the where θd is the design value of material temperature and θcr,d is the design value Simplified assessment of structural elements in terms of their fire resistance is given in tables in STN 730821: 1973, which is currently valid for the assessment of building structures during construction changes. The fire resistance values of building structures are given in particular tables considering building materials and 4.2 Fire resistance assessment of existing fire-separating structures in a model The following model example shows a fire safety solution used in the restoration of a folk house situated in the village of Vel'ké Leváre. The folk house is dated to the Hutterian culture period. It was restored with the intention of preserving its original layout including the original constructions and elements. The building has a combined structural system—the brick external walls, wooden beam ceiling and collar beam truss. It was necessary to optimize the boundary conditions of the given solution so that the consequences of a functional change regarding the current constructions could be minimal. The museum display showing the original culture was situated in the restored space after the original supporting elements, roof covering and original wall and floor surfaces had been replaced or repaired. The external wall is combined stone with bricks. There are wooden ceilings with visible beams supported by a wooden beam. This beam is embedded into the perimeter walls, and its center is supported by a column. The wooden truss has a The building's functional use was changed in terms of fire safety—it became a museum, that is its original residential function was changed into an exhibition one. The fire load increased but only on the first floor. The required value of the time t and Efi,d,t is the design value of fire load effects during the time t. static load of the structures—walls, columns and ceilings [11]. Rfi,d,t ≥ Efi,d,t (2) θd ≥ θcr,d (3) 104 fire safety degree was not changed compared to the original one. The attic spaces used as living rooms serve today only to show the original truss construction. Considering the fire height of 0 m and the combined building's construction, the required fire safety degree is I, that is the same as in the original functional use. The fire resistance requirement for the original load-bearing and fire-separating structures was not changed after the functional change of the restored spaces. The fire resistance requirement for the load-bearing ceiling members and perimeter wall is given according to STN 73082 and is dependent on the average fire load, that is the sum of the accidental and permanent fire load, coefficients of ventilation, flammability factor and use and type of firefighting equipment. In terms of the calculated fire load pv = 66 kg/m2 , the fire resistance requirement for the fire-separated structures is REI 30 (building envelope and roof), and the fire resistance requirement for loadbearing structures of a non-compact ceiling in an assessed fire compartment is R 30. #### 4.3 Fire resistance of the external wall in a model solution The external wall is made of stone and brick and has a variable thickness of 530–630 mm. The required fire resistance for this model solution is REI 30 min. In accordance with the values given in Table 1A STN 730821, the real fire resistance of the perimeter wall is well above the required value (Table 1). As the dimensions of the assessed wooden ceiling members and column in a model solution are different from the members given in the standard, their real fire resistance is calculated according to EN 1996-1-2: 2004 Eurocode 6: Design of masonry structures, Section 1.2 general rules—fire resistance design of masonry structures. In specific cases, the fire resistance of fire-separating structures can be determined by a calculation according to EN 1996-1-2: 2004 Eurocode 6: Design of masonry structures, Section 1.2 general rules—fire resistance design of masonry structures. #### 4.4 Fire resistance of the wooden ceiling in a model solution There is a wooden beamed ceiling above the ground plan in the model example. The ceiling material and structure are visible (see Figure 3). The ceiling beams are supported by a wooden beam. The wooden beam is fastened on the load-bearing peripheral walls and supported by a wooden column (see Figure 4). The real fire resistance for load-bearing and fire-separating structures was calculated according to the methodology given in STN EN 1991-1-2: 2004; the #### Table 1. Fire resistance of the fire-separating structures according to STN 730821 [12]. fire resistance of wooden members was calculated according to STN EN 1995-1-2 (Eurocode 5) depending on their mechanical stress [13, 14]. The real fire resistance for load-bearing and fire-separating structures is primarily dependent on their mechanical load during fire and fire load density caused by building's operation. Determination of fire resistance for structures in a fire compartment depends on the typical fire load density per unit of floor area (qf, d), burning rate coefficient, fire risk coefficients and fire protection coefficients. Estimated fire duration in the assessed fire compartment is determined after considering the influence of structures, ventilation and active firefighting equipment. The fire resistance of wooden members affected by fire (wooden beam ceiling, column and girder) was specified using the effective cross-section method [15]. The methodology is based on the assumption that the first phase of burning wooden elements causes the surface burning and forms a carbonized layer. Such element becomes partially thermo-insulated by further thermal stress, which prolongs its fire resistance. The charring thickness is determined by the fire duration to which the element is exposed and by the charring rate. This interface or the location of the carbonized line in most coniferous and deciduous trees corresponds to the isothermal position of 300°C. After obtaining an effective cross-section, the element is assessed according to [16]. The method of reduced properties works with the residual cross-section (obtained after reading the carbonized layer) taking into account the changed strength and stiffness material properties based on the modified coefficient. In light of this assessment, all wooden load-bearing members in the assessed fire section of the museum met the required fire resistance without additional structural modifications. The assessment of ceiling supporting members in terms of static load at a room temperature is given in Table 2. Table 3 gives the assessment of supporting ceiling members in terms of static and fire load during a standard fire [17]. For material characteristics the following are considered: kfi, coefficient of solid timber, kfi = 1.25; kmod,fi, modification factor for fire, kmod,fi = 1.0; and γM,fi, partial factor for timber in fire, γM,fi = 1.0. For the calculation of charring depth, the following are considered: βn, notional design charring rate under standard fire exposure, βn = 0.8 mm/min (for solid timber); k0, coefficient for non-protected Figure 3. Layout and visible ceiling in a model solution (left) and ground plan with assessed wooden truss (right). 107 d0 = 7 mm. EN 1995-1-2). 1 2 Table 2. Figure 4. decking (right). 5. Solution methods 5.1 Analysis of fire risk meet the required fire resistance value [16]. The average column diameter at its narrowest spot. A Case Study on the Fire Safety in Historic Buildings in Slovakia surfaces, k0 = 1.0; and d0, layer thickness with assumed zero strength and stiffness, [kNm] Ceiling beam 200 250 6.35 0.63 0.53 0.6 Roof girder 300 270 14.64 −0.16 15.8 0.6 Column d = 2302 0.11 1.30 55.50 0.6 where: ηfi—reducing factor for combined load. As simplification it is possible to use the value 0.6 (according to STN Mz,Ed [kNm] NEd1 [kN] ηfi [-] If the restoration of listed buildings is designed, its preparatory phase analyzes the current fire risk in the building. The fire risk analysis examines the current fire Analysis of the current building solution in terms of fire safety—includes assessment of current or planned layouts, flammability of structures and materials, number of risk and the extent of fire-technical and organizational measures [5]. The fire risk assessment can be divided into four phases: The structures assessed in terms of table values given in STN 730821 (see Tables 3 and 5) as well as values determined by a calculation in dependence on the current boundary conditions—static load and material characteristics of wood— Supporting using a wooden beam and column (left) and ceiling wooden beam with a wooden No Member b [mm] h [mm] My,Ed Positive sign (+) means tensile force; negative sign (−) means compression force. Parameters of static load of wooden ceiling supporting members at the room temperature. DOI: http://dx.doi.org/10.5772/intechopen.91241 A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 #### Figure 4. Fire Safety and Management Awareness additional structural modifications. fire resistance of wooden members was calculated according to STN EN 1995-1-2 The real fire resistance for load-bearing and fire-separating structures is primarily dependent on their mechanical load during fire and fire load density caused by building's operation. Determination of fire resistance for structures in a fire compartment depends on the typical fire load density per unit of floor area (qf, d), burning rate coefficient, fire risk coefficients and fire protection coefficients. Estimated fire duration in the assessed fire compartment is determined after considering the influ- The fire resistance of wooden members affected by fire (wooden beam ceiling, column and girder) was specified using the effective cross-section method [15]. The methodology is based on the assumption that the first phase of burning wooden elements causes the surface burning and forms a carbonized layer. Such element becomes partially thermo-insulated by further thermal stress, which prolongs its fire resistance. The charring thickness is determined by the fire duration to which the element is exposed and by the charring rate. This interface or the location of the carbonized line in most coniferous and deciduous trees corresponds to the isothermal position of 300°C. After obtaining an effective cross-section, the element is assessed according to [16]. The method of reduced properties works with the residual cross-section (obtained after reading the carbonized layer) taking into account the changed strength and stiffness material properties based on the modified coefficient. In light of this assessment, all wooden load-bearing members in the assessed fire section of the museum met the required fire resistance without The assessment of ceiling supporting members in terms of static load at a room temperature is given in Table 2. Table 3 gives the assessment of supporting ceiling For material characteristics the following are considered: kfi, coefficient of solid timber, kfi = 1.25; kmod,fi, modification factor for fire, kmod,fi = 1.0; and γM,fi, partial factor for timber in fire, γM,fi = 1.0. For the calculation of charring depth, the following are considered: βn, notional design charring rate under standard fire exposure, βn = 0.8 mm/min (for solid timber); k0, coefficient for non-protected Layout and visible ceiling in a model solution (left) and ground plan with assessed wooden truss (right). members in terms of static and fire load during a standard fire [17]. (Eurocode 5) depending on their mechanical stress [13, 14]. ence of structures, ventilation and active firefighting equipment. 106 Figure 3. Supporting using a wooden beam and column (left) and ceiling wooden beam with a wooden decking (right). where: ηfi—reducing factor for combined load. As simplification it is possible to use the value 0.6 (according to STN EN 1995-1-2). 1 Positive sign (+) means tensile force; negative sign (−) means compression force. 2 The average column diameter at its narrowest spot. #### Table 2. Parameters of static load of wooden ceiling supporting members at the room temperature. surfaces, k0 = 1.0; and d0, layer thickness with assumed zero strength and stiffness, d0 = 7 mm. The structures assessed in terms of table values given in STN 730821 (see Tables 3 and 5) as well as values determined by a calculation in dependence on the current boundary conditions—static load and material characteristics of wood meet the required fire resistance value [16]. #### 5. Solution methods If the restoration of listed buildings is designed, its preparatory phase analyzes the current fire risk in the building. The fire risk analysis examines the current fire risk and the extent of fire-technical and organizational measures [5]. #### 5.1 Analysis of fire risk The fire risk assessment can be divided into four phases: Analysis of the current building solution in terms of fire safety—includes assessment of current or planned layouts, flammability of structures and materials, number of	doab	2025-04-07T03:56:59.089096	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 8 }
006df1b2-f518-4d6b-8d96-484202f13086.9	Table 3. Parameters of static load of wooden ceiling supporting members at the standard fire temperature. 109 equipment, chimneys, etc. working hours. A Case Study on the Fire Safety in Historic Buildings in Slovakia Densities in mega-joules per square meter Pn conversion <sup>730802</sup> Occupancy Mean Percent fractile\* 80 90 95 Dwellings 780 870 920 970 52 40 Hospitals 230 350 440 520 21 20 Hotel rooms 310 400 460 510 24 30 Offices 420 570 670 760 34 40 Shops 600 900 1100 1300 54 90 Museums 300 470 590 720 28 60 Libraries 1500 2250 2550 --- 134 120 Schools 285 360 410 450 22 25 from EK (16,75) 80% fractile Pn value from Table A1 in STN staircases and their location in relation to the center of gravity of evacuated persons, ventilation of staircases and disposition possibilities of their fire separation, evacuate conditions for person and historic articles depending on the building's functional use in fire as well as the accessibility and safety of staircases for firefighters. It also assesses the possibilities of fire spreading in the building's open spaces, e.g. central representative staircases, open galleries, internal atriums, unsealed crawl spaces in ceilings, etc. The analysis determines the construction and division of the building into smaller units—fire compartments, location of fire doors and Comparison of the fire load density values in different occupancies according to the data given in EN 1991-1-2 . The percent fractile is the value that is not exceeded in that percent of the rooms or occupancies. Analysis of the current operational solution in terms of fire prevention—includes an assessment of the building's functional use considering the accidental fire load with regard to the fire resistance of the existing load-bearing structures and the number of persons in terms of the capacity of existing evacuation routes. It also contains assessment of internal organizational measures that should minimize the causes of fire such as regular inspections of electrical installations and appliances, technical Analysis of the current fire detection system—includes an assessment of the function and location of the automatic fire detection system. If there is no such system installed in the building (this is the common situation in historic buildings in Slovakia), it is necessary to verify the organizational measures ensuring fire detection, that is to ask authorized employees to be helpful and use their senses. This includes regular inspections in the building by its guard. If there is no guard in the smaller buildings, the inspection is done by authorized employees at the end of Analysis of the fire equipment availability in case of fire—finds out the location of portable fire extinguishers, their capacity and extinguishing agent. It analyzes the availability of internal firefighting water and wall fire hydrants as well as their position and functionality. It also verifies the location, capacity and functionality of external firefighting water sources, that is external hydrants, water tanks and natural water sources that can be used by fire brigades. It analyzes organizational measures related to fire extinguishing such as staff training, firefighting documentation, identification of emergency routes and access roads. After determining the the way of their closing and risk of fire spreading to adjacent buildings. DOI: http://dx.doi.org/10.5772/intechopen.91241 (MJ/m2 ) Conversion factors: 1 MJ ≈ 0.948 BTU, 1 m2 ≈ 10.8 ft2 \* Table 4. and Table A1 STN 730802. A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 Conversion factors: 1 MJ ≈ 0.948 BTU, 1 m2 ≈ 10.8 ft2 . \ The percent fractile is the value that is not exceeded in that percent of the rooms or occupancies.* #### Table 4. Fire Safety and Management Awareness 108 No 1 2 3 Column d = 2302 1Positive sign (+) means tensile force; negative sign (−) means compression force. 2The average column diameter at its narrowest spot. Table 3. Parameters of static load of wooden ceiling supporting members at the standard fire temperature. 0.07 0.78 33.30 0.142 1.676 1.502 0.16 < 1.0 meets Roof 300 270 8.78 −0.10 9.05 5.118 0.050 0.180 0.23 < 1.0 meets girder Ceiling 200 250 3.81 0.38 0.32 3.454 0.544 0.011 0.15 < 1.0 meets beam Member b [mm] h [mm] My,Ed,fi Mz,Ed.fi NEd,fi σm,y,d,fi σm,z,d,fi σc(t),0,d,fi Assessment bend (bend + tension) + pressure [MPa] [MPa] [MPa] [kN] R30 [kNm] [kN] Comparison of the fire load density values in different occupancies according to the data given in EN 1991-1-2 and Table A1 STN 730802. staircases and their location in relation to the center of gravity of evacuated persons, ventilation of staircases and disposition possibilities of their fire separation, evacuate conditions for person and historic articles depending on the building's functional use in fire as well as the accessibility and safety of staircases for firefighters. It also assesses the possibilities of fire spreading in the building's open spaces, e.g. central representative staircases, open galleries, internal atriums, unsealed crawl spaces in ceilings, etc. The analysis determines the construction and division of the building into smaller units—fire compartments, location of fire doors and the way of their closing and risk of fire spreading to adjacent buildings. Analysis of the current operational solution in terms of fire prevention—includes an assessment of the building's functional use considering the accidental fire load with regard to the fire resistance of the existing load-bearing structures and the number of persons in terms of the capacity of existing evacuation routes. It also contains assessment of internal organizational measures that should minimize the causes of fire such as regular inspections of electrical installations and appliances, technical equipment, chimneys, etc. Analysis of the current fire detection system—includes an assessment of the function and location of the automatic fire detection system. If there is no such system installed in the building (this is the common situation in historic buildings in Slovakia), it is necessary to verify the organizational measures ensuring fire detection, that is to ask authorized employees to be helpful and use their senses. This includes regular inspections in the building by its guard. If there is no guard in the smaller buildings, the inspection is done by authorized employees at the end of working hours. Analysis of the fire equipment availability in case of fire—finds out the location of portable fire extinguishers, their capacity and extinguishing agent. It analyzes the availability of internal firefighting water and wall fire hydrants as well as their position and functionality. It also verifies the location, capacity and functionality of external firefighting water sources, that is external hydrants, water tanks and natural water sources that can be used by fire brigades. It analyzes organizational measures related to fire extinguishing such as staff training, firefighting documentation, identification of emergency routes and access roads. After determining the #### Table 5. 111 A Case Study on the Fire Safety in Historic Buildings in Slovakia firefighting partitions at the required time. • Enable safe evacuation of persons from the building. • Enable effective and safe intervention of fire brigades. reducing the open layouts going through more floors sary to reduce the building's occupancy • Detection of the fire risk resulting from the building's operation roofs and burning other structures. to be changed contains mostly: protection areas fire extinguishers extinguishing equipment. 5.3 Fire compartmentation current fire safety measures in the building, the restoration or functional change is optimized in such a way that the planned alternation would not reduce the current The fire safety in buildings is generally a combination of passive and active measures ensuring the following points for each fire section during the fire: • Retain the carrying capacity and stability of load-bearing structures and • Reduce the development and spread of fire and smoke within the building. • Reduce the spread of fire toward the surrounding buildings through windows, The building solution for a historic building whose original function is planned • Fire compartmentation of the building excluding concentrated fire load and • Fire resistance assessment of existing fire separation structures consider ing the fire risk, fire height and combustibility of load-bearing and fire separation structures including the possible solution for their additional fire • Construction of protected emergency routes if it is possible; if not, it is neces • Ensuring the accessibility of sufficient source of firefighting water and hand • Construction of the safe intervention routes including access roads and boarding If there are some barriers on the access roads to the building such as castle hills or impassable entrance gates, it is necessary to determine a set of construction and fire-technical measures using active elements of fire protection, e.g. stationary fire If it is possible in terms of building's operation, it should always be divided into several smaller fire compartments to minimize fire damage and increase the occu pants' safety during evacuation and fire intervention. If there are no complications DOI: http://dx.doi.org/10.5772/intechopen.91241 building's fire safety. 5.2 Fire safety design Optimization of functional changes in historic buildings in terms of fire protection requirements for original structural elements according to STN 73 0802/2010 and 2015. current fire safety measures in the building, the restoration or functional change is optimized in such a way that the planned alternation would not reduce the current building's fire safety. ### 5.2 Fire safety design Fire Safety and Management Awareness 30/15 110 Original functional use of buildings, Functional usability Fire hazard DFSB Requirements for fire-separating structures in the composite construction unit on the first/last floor STN 730802 Walls REI REI REI EI, EW Ceilings Roof Fire dampers a pv (kg/m2 ) fire height Churches, fh = 0 m Cloister premises, fh ≤ 9 m Galleries Museums Concert halls Libraries Coffee bars Accommodation, apartment buildings Administration Education Gallery Museums Hotels Galleries, museums Hotels Club spaces Kindergartens Hotels, apartment buildings Administration 1.0 1.0 fh—building's fire height; pv—calculated fire load in kg/m2 (the average fire load value of the entire fire compartment); a—coefficient of combustible materials (burning rate) (Table A1 STN 730802); b—coefficient of ventilation efficiency (ventilation rate); REI—time in minutes (minimal time during which the criteria for load, stability and integrity of thermal insulation are met); EI—time in minutes (minimal time during which the criteria for integrity of thermal insulation are met); EW—time in minutes (minimal time during which the criteria for insulation integrity guided by radiation are met); DFSB—degree of fire safety of building structures (expresses the summary of technical requirements for fire-separating structures). Optimization of functional changes in historic buildings in terms of fire protection requirements for original structural elements according to STN 73 0802/2010 and 2015. Table 5. 40 III. 60/45 60/45 45 45/30 30 III. 60/45 60/45 45 45/30 Castles, mansions, fh ≤ 12 m Townhouses, villas, palaces, fh ≤ 12 m 1.0 0.9 1.2 1.1 1.0 1.1 1.0 1.1 0.9 32 III. 60/45 60/45 15 45/30 33 III. 60/45 60/45 45 45/30 30 III. 60/45 60/45 45 45/30 66 III. 60/45 60/45 45 45/30 30 III. 60/45 60/45 45 45/30 66 III. 60/45 60/45 45 45/30 18 II. 45/30 45/30 30 30/30 22 II. 45/30 45/30 30 30/30 40 III. 60/45 60/45 45 45/30 1.2 1.1 1.1 0.7 1.2 1.0 40 III. 60/45 60/45 45 45/30 37 III. 60/45 60/45 45 45/30 84 IV. 90/60 90/60 60 60/D1 33 I. 30/15 30/15 15 30/15 66 I. 30/15 30/15 15 18 I. 30/15 30/15 15 30/15 The fire safety in buildings is generally a combination of passive and active measures ensuring the following points for each fire section during the fire: The building solution for a historic building whose original function is planned to be changed contains mostly: If there are some barriers on the access roads to the building such as castle hills or impassable entrance gates, it is necessary to determine a set of construction and fire-technical measures using active elements of fire protection, e.g. stationary fire extinguishing equipment. #### 5.3 Fire compartmentation If it is possible in terms of building's operation, it should always be divided into several smaller fire compartments to minimize fire damage and increase the occupants' safety during evacuation and fire intervention. If there are no complications during the fire intervention and the fire brigade arrive in the first phase of fire, then there is minor material damage found usually in the fire-affected part of the building. The separate fire compartments always include emergency routes, gathering areas, rooms with a high fire load, warehouses and technical rooms. The fire separation of an existing staircase from adjacent spaces with vertical fire load divides the building into more floors that are simultaneously fire compartments. They reduce the spread of thermal radiation and smoke within the building and relatively safe evacuation [18]. The staircases are separate fire compartments without fire risk; their layout, ventilation and air exchange frequency depend on the time required for evacuation of persons. The separate fire compartments should be all spaces with installations—airconditioning engine rooms, boiler rooms, switch rooms, installation shafts as well as storage areas, deposits, etc. If the spaces are modified for housing, accommodation, hospital or meeting, they must be divided into the fire compartments. Each dwelling unit must be a separate fire compartment; the same is valid for bed sections in hospital, hotel rooms or meeting rooms and museums, exhibition halls, theaters, etc. Any room or fire compartment containing more than 200 people is considered to be a meeting room. There are no exceptions allowed, and it is always necessary to reach an agreement between the fire safety requirements and building conservation. The multistory fire sections require higher fire resistance of building structures than single-story ones, as the fire load is concentrated on the first floor. If fire occurs, it is supposed that the entire building will burn at the same time. The building structures are required to withstand thermal stress without breaking their stability and integrity throughout the fire of the entire building, that is longer than the single-story fire compartment. Finance that are saved by reducing the fireseparating structures such as doors, ceilings, etc. are usually used to ensure the fire resistance of the existing structures if they are composite and combustible. Such solutions absolutely do not respect property protection and safety of persons in the building. If fire damage is to be minimized, the building must be divided into fire compartments. The maximum area of fire compartments depends on the combustibility of the structure, number of floors and coefficient of combustible substances. #### 5.4 Fire risk The fire safety solution in historic buildings whose original function is changed depends on the extent of construction modifications and planned functional use of the original spaces. If the functional use of historic buildings is planned to be changed, the real fire risk related to the planned operation should be taken into account. The fire risk is specified for each fire compartment. Its value depends on the combustibility and heating capacity of materials used in particular spaces depending on their functional use, coefficient of combustible substances, ventilation and active fire safety equipment. It is calculated from the relation: $$\text{qf}, \text{d} = \text{qf}, \text{k.m.} \text{\"\textdegree \textquotesingle}{\text{qf}} \text{\"\textquotesingle}{\text{\"\textquotesingle}{\text{\"\textquotesingle}{\textquotesingle}{\text{\"\textquotesingle}{\textquotesingle}{\textquotesingle}{\text{\"\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}{\textquotesingle}$$ 113 A Case Study on the Fire Safety in Historic Buildings in Slovakia 1991-1-2 and comparison with parameters STN 920201-1). qf,k = \_1 Fire load can be determined by a calculation from relation (5) or from statistical values; examples for selected types of operations are given in Table 1 (source EN ∞ (i. mi. Hui. Mi) (5) Fire load Q in a fire compartment is defined as the total energy that can be released in fire occurrence. One part of the total energy will be used to heat the space (walls and internal gas); the rest of the energy will be released through openings—building elements such as wall and ceiling linings. The building content such as furniture is the fire load. Fire load Q divided by the floor area gives the fire load density qf. Typical fire load density in EC 1 is defined by the equation [10]: Af · ∑<sup>i</sup> where Mi is the mass of material i [kg]; Hui is the net heating value of material i [MJ/kg]; mi is the factor describing combustible properties of material i; Ψi is the factor assessing protected fire load of material i; and Af is the floor area of the fire HuiMi represents the total amount of energy that is contained in material i and released if combustion process is complete. Factor "m" is a non-dimensional factor between 0 and 1 representing combustion efficiency: m = 1 corresponds to complete combustion, and m = 0 if materials do not contribute to fire. The value of m = 0.8 is suggested for standard materials; the value of Hu = 17.5 MJ/kg is sug- Common building designs supposing the use of similar material quantities with the same heating capacity in installations can work with the statistical value of typical fire load density, as defined in EN 1991-1-2; if the designs are done in Slovakia, they follow Table A1 STN 730802. The value of accidental fire load stated in this standard is the weight of wood in kg calculated per unit of the floor area of The functional change of the original spaces changes the fire risk and number of persons. The change of building's fire height, e.g. by roof extension, changes the original building's fire height, fire protection requirements and evacuation plans. An increasing number of persons in the building change the requirements for the capacity and ventilation of emergency routes as well as the fire resistance of fire separation structures. Therefore, it is very important for the investment plan (as for space function and useful floor area extension) to be optimized in such a way that the original boundary conditions would not be changed fundamentally in terms of fire safety and would not require additional significant alternations to the building The fire resistance requirements for building constructions specified in STN 73 0802 are directly dependent on calculated fire load, building's fire height and combustibility of constructions used in a building. It is optimal to prefer opera- containing mostly composite construction systems was changed. This value considers the operational fire load, surface finishes, effect of ventilation and fire-technical equipment. Classrooms, hotel rooms, coffee bars, offices or galleries are classified as pv = (pn + ps) . a . b . c (kg/ m<sup>2</sup> spaces with medium fire load (medium fire development) (see Table 5). Calculated fire load is determined by the relations: all combustible materials in this area. Table 4 shows the data comparison. , whose heating capacity is the same as heating capacity of if the function of restored buildings ) (6) DOI: http://dx.doi.org/10.5772/intechopen.91241 5.5 Fire load compartment [m2 fire compartment in m2 ]. structures affecting their historic value. tions with a calculated fire load up to 50 kg/m<sup>2</sup> gested for wood, resulting in 14 MJ/kg for (m.Hu). where qf, k is the fire load density per floor area unit MJ/m2 ; m is the burning rate coefficient; δq1 is the fire danger; δq2 is the fire danger; and δqn is the function of active fire-protective measures (δqn1–δqn2 automatic fire extinguishers, δqn3– δqn5 automatic fire alarms). The fire load density expresses the probable fire intensity in the fire compartment or its part. #### 5.5 Fire load Fire Safety and Management Awareness the time required for evacuation of persons. as storage areas, deposits, etc. during the fire intervention and the fire brigade arrive in the first phase of fire, then there is minor material damage found usually in the fire-affected part of the building. The separate fire compartments always include emergency routes, gathering The fire separation of an existing staircase from adjacent spaces with vertical fire load divides the building into more floors that are simultaneously fire compartments. They reduce the spread of thermal radiation and smoke within the building and relatively safe evacuation [18]. The staircases are separate fire compartments without fire risk; their layout, ventilation and air exchange frequency depend on The separate fire compartments should be all spaces with installations—airconditioning engine rooms, boiler rooms, switch rooms, installation shafts as well If the spaces are modified for housing, accommodation, hospital or meeting, they must be divided into the fire compartments. Each dwelling unit must be a separate fire compartment; the same is valid for bed sections in hospital, hotel rooms or meeting rooms and museums, exhibition halls, theaters, etc. Any room or fire compartment containing more than 200 people is considered to be a meeting room. There are no exceptions allowed, and it is always necessary to reach an agree- The multistory fire sections require higher fire resistance of building structures than single-story ones, as the fire load is concentrated on the first floor. If fire occurs, it is supposed that the entire building will burn at the same time. The building structures are required to withstand thermal stress without breaking their stability and integrity throughout the fire of the entire building, that is longer than the single-story fire compartment. Finance that are saved by reducing the fireseparating structures such as doors, ceilings, etc. are usually used to ensure the fire resistance of the existing structures if they are composite and combustible. Such solutions absolutely do not respect property protection and safety of persons in the building. If fire damage is to be minimized, the building must be divided into fire compartments. The maximum area of fire compartments depends on the combustibility of the structure, number of floors and coefficient of combustible substances. The fire safety solution in historic buildings whose original function is changed depends on the extent of construction modifications and planned functional use of the original spaces. If the functional use of historic buildings is planned to be changed, the real fire risk related to the planned operation should be taken into account. The fire risk is specified for each fire compartment. Its value depends on the combustibility and heating capacity of materials used in particular spaces depending on their functional use, coefficient of combustible substances, ventila- rate coefficient; δq1 is the fire danger; δq2 is the fire danger; and δqn is the function of active fire-protective measures (δqn1–δqn2 automatic fire extinguishers, δqn3– The fire load density expresses the probable fire intensity in the fire compart- qf,d = qf,k . m . δq1 δq2 δn MJ/ m<sup>2</sup> (4) ; m is the burning tion and active fire safety equipment. It is calculated from the relation: where qf, k is the fire load density per floor area unit MJ/m2 ment between the fire safety requirements and building conservation. areas, rooms with a high fire load, warehouses and technical rooms. 112 δqn5 automatic fire alarms). ment or its part. 5.4 Fire risk Fire load can be determined by a calculation from relation (5) or from statistical values; examples for selected types of operations are given in Table 1 (source EN 1991-1-2 and comparison with parameters STN 920201-1). Fire load Q in a fire compartment is defined as the total energy that can be released in fire occurrence. One part of the total energy will be used to heat the space (walls and internal gas); the rest of the energy will be released through openings—building elements such as wall and ceiling linings. The building content such as furniture is the fire load. Fire load Q divided by the floor area gives the fire load density qf. Typical fire load density in EC 1 is defined by the equation [10]: $$\text{qf.}\,\text{k} = \frac{1}{Af} \cdot \sum\_{i}^{\circ\circ} \left( \psi i.mi.Hui.Mi\right) \tag{5}$$ where Mi is the mass of material i [kg]; Hui is the net heating value of material i [MJ/kg]; mi is the factor describing combustible properties of material i; Ψi is the factor assessing protected fire load of material i; and Af is the floor area of the fire compartment [m2 ]. HuiMi represents the total amount of energy that is contained in material i and released if combustion process is complete. Factor "m" is a non-dimensional factor between 0 and 1 representing combustion efficiency: m = 1 corresponds to complete combustion, and m = 0 if materials do not contribute to fire. The value of m = 0.8 is suggested for standard materials; the value of Hu = 17.5 MJ/kg is suggested for wood, resulting in 14 MJ/kg for (m.Hu). Common building designs supposing the use of similar material quantities with the same heating capacity in installations can work with the statistical value of typical fire load density, as defined in EN 1991-1-2; if the designs are done in Slovakia, they follow Table A1 STN 730802. The value of accidental fire load stated in this standard is the weight of wood in kg calculated per unit of the floor area of fire compartment in m2 , whose heating capacity is the same as heating capacity of all combustible materials in this area. Table 4 shows the data comparison. The functional change of the original spaces changes the fire risk and number of persons. The change of building's fire height, e.g. by roof extension, changes the original building's fire height, fire protection requirements and evacuation plans. An increasing number of persons in the building change the requirements for the capacity and ventilation of emergency routes as well as the fire resistance of fire separation structures. Therefore, it is very important for the investment plan (as for space function and useful floor area extension) to be optimized in such a way that the original boundary conditions would not be changed fundamentally in terms of fire safety and would not require additional significant alternations to the building structures affecting their historic value. The fire resistance requirements for building constructions specified in STN 73 0802 are directly dependent on calculated fire load, building's fire height and combustibility of constructions used in a building. It is optimal to prefer operations with a calculated fire load up to 50 kg/m<sup>2</sup> if the function of restored buildings containing mostly composite construction systems was changed. This value considers the operational fire load, surface finishes, effect of ventilation and fire-technical equipment. Classrooms, hotel rooms, coffee bars, offices or galleries are classified as spaces with medium fire load (medium fire development) (see Table 5). Calculated fire load is determined by the relations: $$\mathbf{p}\mathbf{v} = (\mathbf{p}\mathbf{n} + \mathbf{p}\mathbf{s}) . \mathbf{a}. \mathbf{b}. \mathbf{c} \left(\mathbf{k}\mathbf{g}/\mathbf{m}^2\right) \tag{6}$$ which depends on: pn—accidental fire load from furnishings given in Table A1 STN 730802 [19]; ps—stable fire load from windows, doors, floor and wall coverings given in Table A1 STN 730802; and a—coefficient of combustible materials (burning rate), $$\mathbf{a} = \langle \mathbf{an}.\mathbf{pn} + \mathbf{a}.\mathbf{ps}\rangle / \mathbf{pn} + \mathbf{ps} \tag{7}$$ where an is given in Table A1 STN 730802, as is 0.9 and b is the coefficient of ventilation efficiency (ventilation rate), $$\mathbf{b} = \mathbf{S}. \,\mathrm{k}/\mathbf{S} \mathbf{o}. \,\sqrt{\mathrm{ho}} \tag{8}$$ where S is the floor area of fire compartment; So is the total window area in fire compartment; ho is the average window height in fire compartment; k is the coefficient determined according to Section 4.5.4. STN 730802; and c is the factor of fire safety equipment efficiency, $$\mathbf{c} = \mathbf{c1}.\mathbf{c2}.\mathbf{c3}.\mathbf{c4}\tag{9}$$ where c1 is the coefficient of fire detection (see Table 2 STN 730802); c2 is the coefficient of fire brigade intervention (see Tables 3 and 4 STN 730802); c3 is the coefficient of fixed fire extinguishing system (see Table 5 STN 730802); and c4 is the coefficient of automatic fire sprinklers (see Table 6 STN 730802) Table 5 gives the calculated fire load of a typical fire compartment considering the most common use of space in historic buildings whose function was changed during their use. Model examples considered medium ventilation effect with the coefficient value b = 1. As the most historic buildings do not contain active fire safety equipment, all calculations considered the coefficient value c = 1. Subsequently, DFSB is determined depending on the calculated fire load value, combustibility of structures in the fire compartment and the building's fire height (see Table 8 STN 730802). DFSB expresses the summary of technical requirements for fire-separating structures; required minimum fire resistances of fire-separating structures are taken from Table 12 STN 730802. If building conservation and finance costs are taken into account, it is not possible to carry out every functional change in listed buildings. The building can be classified as unsuitable if fire safety cannot be ensured with reasonable economic and operational costs. The new functional use must not reduce the existing fire safety. In general, listed buildings renovated by using only technical solutions cannot have any functional use. It is optimal for listed buildings to have as low fire risk as possible in terms of fire safety and subsequent fire safety measures [20]. #### 6. Evacuation People evacuated from a burning building are endangered by toxic gases released during combustion, flame, high temperature, smoke and lack of oxygen. The safe evacuation depends on the building's division into fire compartments using fire-separating structures. Their design is based on the assumption that fire will occur in a fire compartment so people present in other fire compartments will not be exposed to fire. The building's division into fire compartments is done in such a way that the life and health loss would be minimal or none. Fire-separating structures in fire compartments should prevent fire and its products from spreading. Separate fire compartments always form protected emergency routes. 115 routes. A Case Study on the Fire Safety in Historic Buildings in Slovakia Fire compartmentation in historic buildings is often limited due to the building conservation. This fact has a major impact on the safe evacuation. Open staircases, galleries and non-solid ceiling structures help fire spreading within such buildings. Thermal radiation, toxic gases and smoke are spread throughout the building. The fire intensity and time are increased by combustible materials in built-in ceilings, columns, staircases, wall facings and insulations of technical installations. This affects the safety and speed of people's movement within the affected fire compart- Safety and fluency of evacuation in historic buildings with original layout and • Open staircases—unprotected emergency routes with limited evacuation time • Partially protected existing narrow spiral or ladder stairs limiting the speed of people's movement that can be used by a limited number of persons during • Limited number of exits leading to an open area through locked doors without • Missing other emergency routes—an absence of other staircases or alternative • Insufficient capacity of escape lanes—inwards opening doors narrowing the These circumstances cause the time for evacuation to be longer, people's safety to The fire development and spreading is a function of time, that is time is crucial for evacuation of people or historic exhibits. Fluent evacuation is conditioned by the number and quality of emergency routes in terms of ventilation, slope, width and number of evacuated persons. Their ventilation and number depend on the building's fire height and the number of evacuated persons. There should be at least two emergency routes available for evacuation in every space; there is significantly better chance of people's survival in spaces directly affected by fire. Evacuated • Missing exits from stairs leading to an open area outside the building automatic opening during fire in single-story buildings escape possibilities through windows, ladders, etc. escape lane and slowing the people's movement speed persons can use the emergency route that is less affected by fire. requirement is ten times the air change per hour. Staircases are used to evacuate people between floors in buildings. According to STN 730802 and the time required for safe evacuation, staircases can be divided into unprotected, partially protected and protected emergency Unprotected routes are open staircases and those located within the fire compartment. Partially protected routes are staircases with fire-separating structures preventing the heat and smoke from spreading and those that are not adequately ventilated. Internally enclosed staircases without natural ventilation are the most common. Protected routes are staircases with fire-separating structures preventing the heat and smoke from spreading and natural or artificial ventilation. Routes of type A with natural or forced ventilation with a maximum evacuation time of 6 min are sufficient for historic buildings with the fire height up to 22.5 m. The ventilation be lower and the risk for firefighting brigade to be higher. DOI: http://dx.doi.org/10.5772/intechopen.91241 ment on unprotected emergency routes. and no other evacuation staircase functional use is often limited by: evacuation #### A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 Fire Safety and Management Awareness ventilation efficiency (ventilation rate), safety equipment efficiency, which depends on: pn—accidental fire load from furnishings given in Table A1 STN 730802 [19]; ps—stable fire load from windows, doors, floor and wall coverings given in Table A1 STN 730802; and a—coefficient of combustible materials (burning rate), where an is given in Table A1 STN 730802, as is 0.9 and b is the coefficient of where S is the floor area of fire compartment; So is the total window area in fire compartment; ho is the average window height in fire compartment; k is the coefficient determined according to Section 4.5.4. STN 730802; and c is the factor of fire where c1 is the coefficient of fire detection (see Table 2 STN 730802); c2 is the coefficient of fire brigade intervention (see Tables 3 and 4 STN 730802); c3 is the coefficient of fixed fire extinguishing system (see Table 5 STN 730802); and c4 is Table 5 gives the calculated fire load of a typical fire compartment considering the most common use of space in historic buildings whose function was changed during their use. Model examples considered medium ventilation effect with the coefficient value b = 1. As the most historic buildings do not contain active fire safety equipment, all calculations considered the coefficient value c = 1. Subsequently, DFSB is determined depending on the calculated fire load value, combustibility of structures in the fire compartment and the building's fire height (see Table 8 STN 730802). DFSB expresses the summary of technical requirements for fire-separating structures; required minimum fire resistances of fire-separating If building conservation and finance costs are taken into account, it is not possible to carry out every functional change in listed buildings. The building can be classified as unsuitable if fire safety cannot be ensured with reasonable economic and operational costs. The new functional use must not reduce the existing fire safety. In general, listed buildings renovated by using only technical solutions cannot have any functional use. It is optimal for listed buildings to have as low fire risk as possible in terms of fire safety and subsequent fire safety measures [20]. People evacuated from a burning building are endangered by toxic gases released during combustion, flame, high temperature, smoke and lack of oxygen. The safe evacuation depends on the building's division into fire compartments using fire-separating structures. Their design is based on the assumption that fire will occur in a fire compartment so people present in other fire compartments will not be exposed to fire. The building's division into fire compartments is done in such a way that the life and health loss would be minimal or none. Fire-separating structures in fire compartments should prevent fire and its products from spreading. Separate fire compartments always form protected emergency routes. the coefficient of automatic fire sprinklers (see Table 6 STN 730802) structures are taken from Table 12 STN 730802. b = S.k/So. a = (an . pn + a . ps)/pn + ps (7) √ho (8) c = c1 . c2 . c3 . c4 (9) 114 6. Evacuation Fire compartmentation in historic buildings is often limited due to the building conservation. This fact has a major impact on the safe evacuation. Open staircases, galleries and non-solid ceiling structures help fire spreading within such buildings. Thermal radiation, toxic gases and smoke are spread throughout the building. The fire intensity and time are increased by combustible materials in built-in ceilings, columns, staircases, wall facings and insulations of technical installations. This affects the safety and speed of people's movement within the affected fire compartment on unprotected emergency routes. Safety and fluency of evacuation in historic buildings with original layout and functional use is often limited by: These circumstances cause the time for evacuation to be longer, people's safety to be lower and the risk for firefighting brigade to be higher. The fire development and spreading is a function of time, that is time is crucial for evacuation of people or historic exhibits. Fluent evacuation is conditioned by the number and quality of emergency routes in terms of ventilation, slope, width and number of evacuated persons. Their ventilation and number depend on the building's fire height and the number of evacuated persons. There should be at least two emergency routes available for evacuation in every space; there is significantly better chance of people's survival in spaces directly affected by fire. Evacuated persons can use the emergency route that is less affected by fire. Staircases are used to evacuate people between floors in buildings. According to STN 730802 and the time required for safe evacuation, staircases can be divided into unprotected, partially protected and protected emergency routes. Unprotected routes are open staircases and those located within the fire compartment. Partially protected routes are staircases with fire-separating structures preventing the heat and smoke from spreading and those that are not adequately ventilated. Internally enclosed staircases without natural ventilation are the most common. Protected routes are staircases with fire-separating structures preventing the heat and smoke from spreading and natural or artificial ventilation. Routes of type A with natural or forced ventilation with a maximum evacuation time of 6 min are sufficient for historic buildings with the fire height up to 22.5 m. The ventilation requirement is ten times the air change per hour. #### 6.1 Solution example of a model building's restoration in terms of evacuation The change of building's functional use and fire load usually results in an increasing number of persons in building compared to the original solution. The evacuation conditions are also changed if the building's fire height is changed, e.g. due to the addition of one or two floors into the attic. Since both cases fundamentally affect the evacuation conditions, it is necessary to check the original emergency routes and modify so that they would be suitable for the new number of evacuated persons or longer emergency route. Here is the solution example of a model building. The new owner of a manor house changed the building's functional use and fire height by adding a floor into the unused attic space. The manor house is a typical baroque building with a U-shaped ground plan. The manor house had originally three above-ground floors with a mansard roof. The building once served as a residence of a noble family. After restoration, it will serve as a hotel. There are social spaces containing inner halls, smaller salons, restaurants, kitchen and sanitary operational background on the ground floor and first floor. Hotel rooms with technical and operational facilities are located on the upper floors (see Figure 5). Each side wing contains one double-wing staircase that was originally open and classified as unprotected at the time of evacuation. Designed building's alternation by hotel rooms built in the attic changed building's fire height and extended staircases beyond the allowable dimensional limits defined in Table 16 STN 730802. It was necessary to alter existing staircases in the side wings. The staircases on each floor were fire-separated from the other fire-loaded spaces and ventilated through existing windows facing the inner courtyard (see Figure 5). Figure 5. Emergency routes on the first floor leading to an open area in a model solution of the restored manor house. 117 Thanks A Case Study on the Fire Safety in Historic Buildings in Slovakia To achieve higher fire safety in historic buildings whose functional use was changed, it is recommended to optimize the fire risk considering combustibility of building structures and building's fire height. Authors J. Li, H. Li, B. Zhou and X. Wang in their work "Investigation and Statistical Analysis of Fire Load of 83 Historic Buildings in Beijing" analyzed the fire load in timber historic buildings where the primary requirement of the restoration was the optimization of acciden- The building should be divided into fire compartments if it is acceptable in terms of the building conservation. If it is possible, another emergency route with direct ventilation should be created. This route would also serve for firefighting intervention. The large roof spaces should be divided into smaller units using fireseparating walls overlapping the roof by at least 300 mm. An accidental fire loads should be excluded from the attic space. All attic entrances should be provided with self-closing fire doors. Interventions into the original floors should be reduced. Hidden cavities in the floors should be analyzed in the project documentation due to the load-bearing capacity during the fire intervention as well as in terms of the occurrence of hidden fire caused by short circuits in electrical installations. All cable entries, pipes and anchoring of heavy chandeliers through ceilings should be carefully fire-sealed. The copper roofing on wooden decking or wooden shingles should be replaced with non-combustible roofing made of burnt tiles or slate—see an example of the castle of Krásna Hôrka. The baroque buildings on the Svatá Hora near the town of Příbram in the Czech Republic underwent a similar restoration after a large fire in 1798. The fire affected buildings' wooden roofs as in Krásna Hôrka. The original wooden shingles were replaced by ceramic tiles after fire. The roof spaces in buildings of significant historic importance should be equipped with an automatic fire alarm system, ideally supplemented with an automatic fire extinguishing system. An example of such solution is the protection of the supporting truss members in St. Vitus Cathedral in Prague, Czech Republic. There is an electrical fire alarm and automatic sprinkler fire extinguishing system installed in The spaces containing visible combustible load-bearing and fire-separating structures should be equipped with an automatic fire alarm system. Water sources that can be used for fire extinguishing should be sufficient and located near the building. Accessibility of water sources is often complicated in historic buildings. Access roads should be verified and optimized within natural possibilities. It is important for the building's operation and its fire safety to have functional firefighting equipment and fire-trained staff so that the risks associated with building's The work is published with the financial support of the project VEGA 1/0248/19. The current fire documentation should be elaborated and updated so as to provide sufficient information on the evacuation plans for persons and exhibits, building's structural design, firefighting water sources and technical condition of capacity equipped with One of the possibilities is the use of water tanks with 10 m3 restoration and maintenance can be minimized [22–24]. The work presents knowledge from the author's design practice. DOI: http://dx.doi.org/10.5772/intechopen.91241 7. Conclusions tal fire load [20]. its roof space. a 50-m-long fire hose [21]. access and emergency roads. #### 7. Conclusions Fire Safety and Management Awareness evacuated persons or longer emergency route. ties are located on the upper floors (see Figure 5). existing windows facing the inner courtyard (see Figure 5). 6.1 Solution example of a model building's restoration in terms of evacuation The change of building's functional use and fire load usually results in an increasing number of persons in building compared to the original solution. The evacuation conditions are also changed if the building's fire height is changed, e.g. due to the addition of one or two floors into the attic. Since both cases fundamentally affect the evacuation conditions, it is necessary to check the original emergency routes and modify so that they would be suitable for the new number of Here is the solution example of a model building. The new owner of a manor house changed the building's functional use and fire height by adding a floor into the unused attic space. The manor house is a typical baroque building with a U-shaped ground plan. The manor house had originally three above-ground floors with a mansard roof. The building once served as a residence of a noble family. After restoration, it will serve as a hotel. There are social spaces containing inner halls, smaller salons, restaurants, kitchen and sanitary operational background on the ground floor and first floor. Hotel rooms with technical and operational facili- Each side wing contains one double-wing staircase that was originally open and classified as unprotected at the time of evacuation. Designed building's alternation by hotel rooms built in the attic changed building's fire height and extended staircases beyond the allowable dimensional limits defined in Table 16 STN 730802. It was necessary to alter existing staircases in the side wings. The staircases on each floor were fire-separated from the other fire-loaded spaces and ventilated through Emergency routes on the first floor leading to an open area in a model solution of the restored manor house. 116 Figure 5. To achieve higher fire safety in historic buildings whose functional use was changed, it is recommended to optimize the fire risk considering combustibility of building structures and building's fire height. Authors J. Li, H. Li, B. Zhou and X. Wang in their work "Investigation and Statistical Analysis of Fire Load of 83 Historic Buildings in Beijing" analyzed the fire load in timber historic buildings where the primary requirement of the restoration was the optimization of accidental fire load [20]. The building should be divided into fire compartments if it is acceptable in terms of the building conservation. If it is possible, another emergency route with direct ventilation should be created. This route would also serve for firefighting intervention. The large roof spaces should be divided into smaller units using fireseparating walls overlapping the roof by at least 300 mm. An accidental fire loads should be excluded from the attic space. All attic entrances should be provided with self-closing fire doors. Interventions into the original floors should be reduced. Hidden cavities in the floors should be analyzed in the project documentation due to the load-bearing capacity during the fire intervention as well as in terms of the occurrence of hidden fire caused by short circuits in electrical installations. All cable entries, pipes and anchoring of heavy chandeliers through ceilings should be carefully fire-sealed. The copper roofing on wooden decking or wooden shingles should be replaced with non-combustible roofing made of burnt tiles or slate—see an example of the castle of Krásna Hôrka. The baroque buildings on the Svatá Hora near the town of Příbram in the Czech Republic underwent a similar restoration after a large fire in 1798. The fire affected buildings' wooden roofs as in Krásna Hôrka. The original wooden shingles were replaced by ceramic tiles after fire. The roof spaces in buildings of significant historic importance should be equipped with an automatic fire alarm system, ideally supplemented with an automatic fire extinguishing system. An example of such solution is the protection of the supporting truss members in St. Vitus Cathedral in Prague, Czech Republic. There is an electrical fire alarm and automatic sprinkler fire extinguishing system installed in its roof space. The spaces containing visible combustible load-bearing and fire-separating structures should be equipped with an automatic fire alarm system. Water sources that can be used for fire extinguishing should be sufficient and located near the building. Accessibility of water sources is often complicated in historic buildings. One of the possibilities is the use of water tanks with 10 m3 capacity equipped with a 50-m-long fire hose [21]. Access roads should be verified and optimized within natural possibilities. It is important for the building's operation and its fire safety to have functional firefighting equipment and fire-trained staff so that the risks associated with building's restoration and maintenance can be minimized [22–24]. The current fire documentation should be elaborated and updated so as to provide sufficient information on the evacuation plans for persons and exhibits, building's structural design, firefighting water sources and technical condition of access and emergency roads. #### Thanks The work is published with the financial support of the project VEGA 1/0248/19. The work presents knowledge from the author's design practice. Fire Safety and Management Awareness ### Author details Agnes Iringová Faculty of Civil Engineering, University of Žilina, Žilina, Slovakia \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 119* A Case Study on the Fire Safety in Historic Buildings in Slovakia Praha: ČVUT Publishing House; 2005. [11] Iringova A, Idunk R. Assessment and usability of historic trusses in terms of fire protection—A case study. International Wood Products Journal. [12] STN 730821. Fire protection of buildings. In: Fire Resistance of Engineering Structures. Praha: [13] STN EN 1995-1-1 + A1. Eurocode 5. Design of Wooden Structures—Part 1-1. Generally—General Rules and Rules for Buildings. Bratislava: SUTN; 2008 [14] STN EN 1995-1-2. Eurocode 5. In: Design of Wooden Structures—Part 1-2. General Rules—Design of Structures for Fire Effect. Bratislava: SUTN; 2008 [15] Vassart O, Zhao B, Cajot LG, [16] Gašpercová S, Makovická L, Kostelanský T. Assessment of fire protection in the castle of Trenčín. Available from: https://stavba.tzb-info. [17] STN EN 338. Constructional wood. In: Strength Classes. Bratislava: SUTN; [18] Emery S. Emergency Plans for Heritage Buildings and Collections. London: English Heritage; 2011 [19] STN 73 0802. Structural fire protection. In: Common Regulations. [20] Li J, Li H, Zhou B, Wang X. Investigation and statistical analysis Bratislava: SUTN; 2010 cz/historicke-stavby/17908 Robert F, Meyer U, Frangi A. Eurocodes: Background applications structural fire design. In: Report EUR26698 EN. European Union; 2014. ISSN 336 p. ISBN 80-0103157-8 2017;8(2):80-87 UNMZ; 1973 1831-9424 2010 DOI: http://dx.doi.org/10.5772/intechopen.91241 [1] Stewart K, Haire S. Fire Safety Management in Traditional Buildings: [2] Egri J. Fire in the castle of Krásna Hôrka. In: PYROMEETING—Fire Protection of Historical Monuments. [3] Firenet: History of Fire Safety (online). 2009. Available from: http:// www.fire.org.uk/history-of-fire-safety. [4] STN 73 0834. Fire safety of buildings. In: Changes in Buildings. Bratislava: [5] Svoboda P, Polatova E. Methodology for fire protection of accessible monuments. In: Proceedings of the Bridges to Fire Protection of Cultural Monuments, Prague. 2015. pp. 32-37 [6] Caston P. Historic roof trusses between 1500 and 1700 in German speaking Central Europe: Documentation, analysis, and development. In: Second International Congress on Construction History, Queens' College, Cambridge University, March 29 to April 04. 2006. [7] STN EN 1991-1-1. Eurocode 1. In: Load of Structures—Part 1-1. Bratislava: [8] STN EN 1991-1-3. Eurocode 1. In: Load of Structures—Part 1-3. Bratislava: [9] STN EN 1991-1-4. Eurocode 1: Load of Structures—Part 1-4. Bratislava: [10] Wald F et al. Calculation of Fire Resistance of Building Structures. Part 1. Principles and Practise. Edinburgh: Historic Scotland; 2010. ISBN 978-1-84917-035-2 Brno. 2013 References SUTN; 2010 pp. 579-597 SUTN; 2007 SUTN; 2007 SUTN; 2007 html A Case Study on the Fire Safety in Historic Buildings in Slovakia DOI: http://dx.doi.org/10.5772/intechopen.91241 #### References Fire Safety and Management Awareness 118 Author details Agnes Iringová Faculty of Civil Engineering, University of Žilina, Žilina, Slovakia © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, \Address all correspondence to: [email protected] provided the original work is properly cited. [1] Stewart K, Haire S. Fire Safety Management in Traditional Buildings: Part 1. Principles and Practise. Edinburgh: Historic Scotland; 2010. ISBN 978-1-84917-035-2 [2] Egri J. Fire in the castle of Krásna Hôrka. In: PYROMEETING—Fire Protection of Historical Monuments. Brno. 2013 [3] Firenet: History of Fire Safety (online). 2009. Available from: http:// www.fire.org.uk/history-of-fire-safety. html [4] STN 73 0834. Fire safety of buildings. In: Changes in Buildings. Bratislava: SUTN; 2010 [5] Svoboda P, Polatova E. Methodology for fire protection of accessible monuments. In: Proceedings of the Bridges to Fire Protection of Cultural Monuments, Prague. 2015. pp. 32-37 [6] Caston P. Historic roof trusses between 1500 and 1700 in German speaking Central Europe: Documentation, analysis, and development. In: Second International Congress on Construction History, Queens' College, Cambridge University, March 29 to April 04. 2006. pp. 579-597 [7] STN EN 1991-1-1. Eurocode 1. In: Load of Structures—Part 1-1. Bratislava: SUTN; 2007 [8] STN EN 1991-1-3. Eurocode 1. In: Load of Structures—Part 1-3. Bratislava: SUTN; 2007 [9] STN EN 1991-1-4. Eurocode 1: Load of Structures—Part 1-4. Bratislava: SUTN; 2007 [10] Wald F et al. Calculation of Fire Resistance of Building Structures. Praha: ČVUT Publishing House; 2005. 336 p. ISBN 80-0103157-8 [11] Iringova A, Idunk R. Assessment and usability of historic trusses in terms of fire protection—A case study. International Wood Products Journal. 2017;8(2):80-87 [12] STN 730821. Fire protection of buildings. In: Fire Resistance of Engineering Structures. Praha: UNMZ; 1973 [13] STN EN 1995-1-1 + A1. Eurocode 5. Design of Wooden Structures—Part 1-1. Generally—General Rules and Rules for Buildings. Bratislava: SUTN; 2008 [14] STN EN 1995-1-2. Eurocode 5. In: Design of Wooden Structures—Part 1-2. General Rules—Design of Structures for Fire Effect. Bratislava: SUTN; 2008 [15] Vassart O, Zhao B, Cajot LG, Robert F, Meyer U, Frangi A. Eurocodes: Background applications structural fire design. In: Report EUR26698 EN. European Union; 2014. ISSN 1831-9424 [16] Gašpercová S, Makovická L, Kostelanský T. Assessment of fire protection in the castle of Trenčín. Available from: https://stavba.tzb-info. cz/historicke-stavby/17908 [17] STN EN 338. Constructional wood. In: Strength Classes. Bratislava: SUTN; 2010 [18] Emery S. Emergency Plans for Heritage Buildings and Collections. London: English Heritage; 2011 [19] STN 73 0802. Structural fire protection. In: Common Regulations. Bratislava: SUTN; 2010 [20] Li J, Li H, Zhou B, Wang X. Investigation and statistical analysis of fire load of 83 historic buildings in Beijing. International Journal of Architectural Heritage. 2018 [21] Karlsen E. Fire Protection of Norwegian Cultural Heritage. Norway: Directorate for Cultural Heritage (Riksantikvaren). Available from: http:// www.arcchip.cz/w04/w04\_karlsen.pdf [22] Ditlev J, Orrainen M. Managing fire safety in historical buildings. In: CFPA-E Guideline No. 30: 2013. F Copenhagen: CFPA Europe; 2013 [23] German Insurance Association. Brandschutz in historischen Gebäuden. Empfehlungen zur Schadenverhütung (VdS 2171). 2008-2012 [24] Jensen G, Cowi AS. Manual Fire Extinguishing Equipment for Protection of Heritage. Norway: Riksantikvaren the Norwegian Directorate for Cultural Heritage Historic Scotland: Technical Conservation, Research and Edication Group; 2006. ISBN: 82-7574-039-8 121* Chapter 7 Abstract carried out. 1. Introduction Gas Industry Rachid Nait-Said and Fatiha Zidani Keywords: BLEVE effects, CFD, FDS, fireball, LES, QRA BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case of an Algeria This chapter presents the numerical modeling of the BLEVE (Boiling Liquid Expanding Vapor Explosion) thermal effects. The goal is to highlight the possibility to use numerical data in order to estimate the potential damage that would be caused by the BLEVE, based on quantitative risk analysis (QRA). The numerical modeling is carried out using the computational fluid dynamics (CFD) code Fire Dynamics Simulator (FDS) version 6. The BLEVE is defined as a fireball, and in this work, its source is modeled as a vertical release of hot fuel in a short time. Moreover, the fireball dynamics is based on a single-step combustion using an eddy dissipation concept (EDC) model coupled with the default large eddy simulation (LES) turbulence model. Fireball characteristics (diameter, height, heat flux and lifetime) issued from a large-scale experiment are used to demonstrate the ability of FDS to simulate the various steps of the BLEVE phenomenon from ignition up to total burnout. A comparison between BAM (Bundesanstalt für Materialforschung und –prüfung, Allemagne) experiment data and predictions highlights the ability of FDS to model BLEVE effects. From this, a numerical study of the thermal effects of BLEVE in the largest gas field in Algeria was After the industrial revolution of the nineteenth century, the world has experienced significant growth in new technologies embedded in the process industry such as gas processing, manufacture of transportation means, etc. In these installations, several fuel elements are present and require special attention in order to avoid accidents whose consequences have severe impacts on people, equipment, and environment. The most common accidents encountered in the chemical and petrochemical process industry are fires, explosions, and toxic releases. Considering the number of existing and future installations, the consequences of Brady Manescau, Khaled Chetehouna, Ilyas Sellami, #### Chapter 7 Fire Safety and Management Awareness of fire load of 83 historic buildings in Beijing. International Journal of Architectural Heritage. 2018 [21] Karlsen E. Fire Protection of Norwegian Cultural Heritage. Norway: Directorate for Cultural Heritage CFPA Europe; 2013 (VdS 2171). 2008-2012 82-7574-039-8 (Riksantikvaren). Available from: http:// www.arcchip.cz/w04/w04\_karlsen.pdf [22] Ditlev J, Orrainen M. Managing fire safety in historical buildings. In: CFPA-E Guideline No. 30: 2013. F Copenhagen: [23] German Insurance Association. Brandschutz in historischen Gebäuden. Empfehlungen zur Schadenverhütung [24] Jensen G, Cowi AS. Manual Fire Extinguishing Equipment for Protection of Heritage. Norway: Riksantikvaren the Norwegian Directorate for Cultural Heritage Historic Scotland: Technical Conservation, Research and Edication Group; 2006. ISBN: 120 ## BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case of an Algeria Gas Industry Brady Manescau, Khaled Chetehouna, Ilyas Sellami, Rachid Nait-Said and Fatiha Zidani ## Abstract This chapter presents the numerical modeling of the BLEVE (Boiling Liquid Expanding Vapor Explosion) thermal effects. The goal is to highlight the possibility to use numerical data in order to estimate the potential damage that would be caused by the BLEVE, based on quantitative risk analysis (QRA). The numerical modeling is carried out using the computational fluid dynamics (CFD) code Fire Dynamics Simulator (FDS) version 6. The BLEVE is defined as a fireball, and in this work, its source is modeled as a vertical release of hot fuel in a short time. Moreover, the fireball dynamics is based on a single-step combustion using an eddy dissipation concept (EDC) model coupled with the default large eddy simulation (LES) turbulence model. Fireball characteristics (diameter, height, heat flux and lifetime) issued from a large-scale experiment are used to demonstrate the ability of FDS to simulate the various steps of the BLEVE phenomenon from ignition up to total burnout. A comparison between BAM (Bundesanstalt für Materialforschung und –prüfung, Allemagne) experiment data and predictions highlights the ability of FDS to model BLEVE effects. From this, a numerical study of the thermal effects of BLEVE in the largest gas field in Algeria was carried out. Keywords: BLEVE effects, CFD, FDS, fireball, LES, QRA #### 1. Introduction After the industrial revolution of the nineteenth century, the world has experienced significant growth in new technologies embedded in the process industry such as gas processing, manufacture of transportation means, etc. In these installations, several fuel elements are present and require special attention in order to avoid accidents whose consequences have severe impacts on people, equipment, and environment. The most common accidents encountered in the chemical and petrochemical process industry are fires, explosions, and toxic releases. Considering the number of existing and future installations, the consequences of these types of accidents remain a major concern for decision-makers, industrial experts, and fire safety analysts. In the context of defining an accurate assessment of the safety of industrial facilities, risk analysts often use quantitative risk analysis (QRA) [1]. It is an analysis method that makes it possible to understand and quantify the consequences of accidental phenomena (thermal radiation, overpressure, toxicity dose). Among the accidental phenomena most observed in the process industry is the boiling liquid expanding vapor explosion (BLEVE). It corresponds to a violent vaporization of explosive nature following the rupture (loss of confinement) of a tank containing a liquid at a temperature significantly higher than its normal boiling point at atmospheric pressure [2]. Between 1940 and 2005, the different BLEVEs listed have cost more than 1000 lives and have injured more than 10,000 people in addition to harming property worth billions of dollars [3]. In addition to human lives and material goods, BLEVE has hazardous effects on the environment; it can release dangerous substances likely to attack the environment. Considering this, it is important to estimate the potential damage that would be caused by such an explosion. In this context, several studies have been conducted to analyze the BLEVE mechanisms. Thermal radiation hazards associated with liquefied petroleum gas (LPG) releases from pressurized storage were studied by Roberts [4]. He established correlations allowing to obtain the fireball characteristic parameters from the fuel mass (diameter, lifetime, and heat flux). From these mathematical laws, Crocker and Napier [5] evaluated fire and explosion hazards of LPG. They showed that these models overestimate the risks associated with jet fires, fireballs, and BLEVE blast effects. Prugh [6], in his part, studied the effects of fuel type and fuel quantity on fireball diameter, duration, and energy and the relationships between fireball energy, distance from the fireball, and consequences of personnel and property exposure. Roberts et al. [7] presented results from a series of experimental tests performed by the Health and Safety Laboratory in the context of JIVE project (hazards consequences of jet fire interaction with vessels containing pressurized liquids). During these tests, several propane tanks were exposed to fires. They allowed to identify the conditions of temperature and rupture pressure, failure mode, as well as the fireball characteristics. In a study conducted by Abbasi et al. [3], the mechanism, the causes, the consequences, the hand calculation methods, and the preventive strategies associated with BLEVEs were presented in an excellent review. Based on medium-scale experimental tests, Birk et al. [8] concluded that the liquid part does not contribute to the generation of shock waves. They proposed a model based on the TNO model that uses the vapor part to calculate the expansion energy. Other works like Bubbico and Marchini [9] and Chen et al. [10] give information on the fact that BLEVE evolution process is characterized by two-phase flow with an overpressure effect. In works cited above, there are empirical and semiempirical approaches which provide data highlighting the characteristics of BLEVE. However, these approaches are not very satisfactory because they usually include an experimentally adjusted reduction factor and mostly overestimate the BLEVE effects [11–13]. Furthermore, they do not consider the effect of buildings, obstructions, and topography for specific facilities. In addition, the data provided by these approaches may not ensure enough repertory for conducting an in-depth QRA. In order to overcome the empirical approach limitations, it is necessary to use the computational fluid dynamics (CFD) modeling which appears as a powerful complementary tool for experimental and theoretical studies. Considering the complexity of the BLEVE phenomenon process, current published CFD simulation studies [14–19] focus only on certain BLEVE aspects, such as fireball formation, 123 BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… ing turbulence, combustion process, heat transfer, and geometry. without considering vessel disintegration. Indeed, with a sufficiently fine numerical resolution, it is possible to carry out simulations of explosion phenomena consider- Among the numerical studies on the BLEVE, Yakush and Makhviladze [14] compared the fireball lifetime predictions from two turbulence models (based on RANS and LES approach) and the fireball lifetime obtained by the experimental correlation of Roper et al. [15]. The simulations were performed by the CFD code FDS from NIST version 4. They showed that the simulation using LES model better predicts fireball dynamics than the simulation using RANS model. Other simulations were made using the CFD code FDS [16–18]. The FDS validation was carried out using the experimental data such as the BAM BLEVE experiment [19]. They evaluated the code capabilities to simulate the fireball characteristics (diameter, lifetime, flame dynamics, and structure). In addition to FDS, other CFD codes are used to simulate fireball characteristics such as OpenFOAM, Ansys CFX, etc. Indeed, Mishra et al. [20] performed a CFD investigation on a peroxy-fuel BLEVE using the CFD commercial code Ansys CFX, and Shelke et al. [21] used the OpenFOAM CFD code. They highlighted the abilities of these CFD codes to predict the reactive flows present in a fireball In this chapter, in addition to evaluating the capability of the CFD code FDS to predict the BLEVE characteristics, an evaluation of the BLEVE thermal effects on a real gas processing plant is presented. The evaluation of the CFD code is made using data obtained from empirical correlations and large-scale experimental data issued from the literature. The calculations are carried out using the FDS code In this context, an overview of the BLEVE phenomenon is presented in the second part of the chapter. In the third part, the capability of FDS to predict BLEVE characteristics is presented in comparison with experimental data. In the fourth part, the BLEVE thermal effects on a real case study are illustrated to finish with BLEVE is described as a violent explosive vaporization resulting from the rupture of a tank containing a liquid at a temperature significantly above its boiling BLEVE can occur with any liquid, flammable or not, when heated and pressurized into a closed container. Two types of BLEVE can be distinguished, cold BLEVE and hot BLEVE, depending on the temperature at which the rupture of the In this illustration, the hot BLEVE with a flammable liquid is studied. The BLEVE explosion of hydrocarbon fuels (e.g., LPG, LNG, etc.) is characterized by the formation of fireball and the release of intense thermal radiation in a In the focus to characterize the BLEVE phenomenon with enough accuracy, it is important to define an experimental setup with a fine and controlled DOI: http://dx.doi.org/10.5772/intechopen.92990 conclusions and perspectives in the last part. 2.2 Description of the different BLEVE tests 2. BLEVE presentation point at atmospheric pressure. 2.1 BLEVE definition enclosure occurs. short time. such as BLEVE. version 6. #### BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 without considering vessel disintegration. Indeed, with a sufficiently fine numerical resolution, it is possible to carry out simulations of explosion phenomena considering turbulence, combustion process, heat transfer, and geometry. Among the numerical studies on the BLEVE, Yakush and Makhviladze [14] compared the fireball lifetime predictions from two turbulence models (based on RANS and LES approach) and the fireball lifetime obtained by the experimental correlation of Roper et al. [15]. The simulations were performed by the CFD code FDS from NIST version 4. They showed that the simulation using LES model better predicts fireball dynamics than the simulation using RANS model. Other simulations were made using the CFD code FDS [16–18]. The FDS validation was carried out using the experimental data such as the BAM BLEVE experiment [19]. They evaluated the code capabilities to simulate the fireball characteristics (diameter, lifetime, flame dynamics, and structure). In addition to FDS, other CFD codes are used to simulate fireball characteristics such as OpenFOAM, Ansys CFX, etc. Indeed, Mishra et al. [20] performed a CFD investigation on a peroxy-fuel BLEVE using the CFD commercial code Ansys CFX, and Shelke et al. [21] used the OpenFOAM CFD code. They highlighted the abilities of these CFD codes to predict the reactive flows present in a fireball such as BLEVE. In this chapter, in addition to evaluating the capability of the CFD code FDS to predict the BLEVE characteristics, an evaluation of the BLEVE thermal effects on a real gas processing plant is presented. The evaluation of the CFD code is made using data obtained from empirical correlations and large-scale experimental data issued from the literature. The calculations are carried out using the FDS code version 6. In this context, an overview of the BLEVE phenomenon is presented in the second part of the chapter. In the third part, the capability of FDS to predict BLEVE characteristics is presented in comparison with experimental data. In the fourth part, the BLEVE thermal effects on a real case study are illustrated to finish with conclusions and perspectives in the last part. #### 2. BLEVE presentation #### 2.1 BLEVE definition Fire Safety and Management Awareness experts, and fire safety analysts. these types of accidents remain a major concern for decision-makers, industrial In the context of defining an accurate assessment of the safety of industrial facilities, risk analysts often use quantitative risk analysis (QRA) [1]. It is an analysis method that makes it possible to understand and quantify the consequences of accidental phenomena (thermal radiation, overpressure, toxicity dose). Among the accidental phenomena most observed in the process industry is the boiling liquid expanding vapor explosion (BLEVE). It corresponds to a violent vaporization of explosive nature following the rupture (loss of confinement) of a tank containing a liquid at a temperature significantly higher than its normal boiling point at atmospheric pressure [2]. Between 1940 and 2005, the different BLEVEs listed have cost more than 1000 lives and have injured more than 10,000 people in addition to harming property worth billions of dollars [3]. In addition to human lives and material goods, BLEVE has hazardous effects on the environment; it can release dangerous substances likely to attack the environment. Considering this, it is important to estimate the potential damage that would be caused by such an explosion. In this context, several studies have been conducted to analyze the BLEVE mechanisms. Thermal radiation hazards associated with liquefied petroleum gas (LPG) releases from pressurized storage were studied by Roberts [4]. He established correlations allowing to obtain the fireball characteristic parameters from the fuel mass (diameter, lifetime, and heat flux). From these mathematical laws, Crocker and Napier [5] evaluated fire and explosion hazards of LPG. They showed that these models overestimate the risks associated with jet fires, fireballs, and BLEVE blast effects. Prugh [6], in his part, studied the effects of fuel type and fuel quantity on fireball diameter, duration, and energy and the relationships between fireball energy, distance from the fireball, and consequences of personnel and property exposure. enough repertory for conducting an in-depth QRA. Roberts et al. [7] presented results from a series of experimental tests performed by the Health and Safety Laboratory in the context of JIVE project (hazards consequences of jet fire interaction with vessels containing pressurized liquids). During these tests, several propane tanks were exposed to fires. They allowed to identify the conditions of temperature and rupture pressure, failure mode, as well as the fireball characteristics. In a study conducted by Abbasi et al. [3], the mechanism, the causes, the consequences, the hand calculation methods, and the preventive strategies associated with BLEVEs were presented in an excellent review. Based on medium-scale experimental tests, Birk et al. [8] concluded that the liquid part does not contribute to the generation of shock waves. They proposed a model based on the TNO model that uses the vapor part to calculate the expansion energy. Other works like Bubbico and Marchini [9] and Chen et al. [10] give information on the fact that BLEVE evolution process is characterized by two-phase flow with an In works cited above, there are empirical and semiempirical approaches which provide data highlighting the characteristics of BLEVE. However, these approaches are not very satisfactory because they usually include an experimentally adjusted reduction factor and mostly overestimate the BLEVE effects [11–13]. Furthermore, they do not consider the effect of buildings, obstructions, and topography for specific facilities. In addition, the data provided by these approaches may not ensure In order to overcome the empirical approach limitations, it is necessary to use the computational fluid dynamics (CFD) modeling which appears as a powerful complementary tool for experimental and theoretical studies. Considering the complexity of the BLEVE phenomenon process, current published CFD simulation studies [14–19] focus only on certain BLEVE aspects, such as fireball formation, 122 overpressure effect. BLEVE is described as a violent explosive vaporization resulting from the rupture of a tank containing a liquid at a temperature significantly above its boiling point at atmospheric pressure. BLEVE can occur with any liquid, flammable or not, when heated and pressurized into a closed container. Two types of BLEVE can be distinguished, cold BLEVE and hot BLEVE, depending on the temperature at which the rupture of the enclosure occurs. In this illustration, the hot BLEVE with a flammable liquid is studied. The BLEVE explosion of hydrocarbon fuels (e.g., LPG, LNG, etc.) is characterized by the formation of fireball and the release of intense thermal radiation in a short time. #### 2.2 Description of the different BLEVE tests In the focus to characterize the BLEVE phenomenon with enough accuracy, it is important to define an experimental setup with a fine and controlled instrumentation. However, the current measurement instruments do not allow the proper acquisition of results during a BLEVE test due to its magnitude. In addition, the high cost of this type of test and considering respect for the environment, there are few experimental tests that deal with this kind of phenomenon. In the literature [19, 22], there are large-scale experiment tests: the BAM test (Bundesanstalt für Materialforschung und –prüfung, Allemagne), the British Gas experiments, and the JIVE tests (hazards consequences of jet fire interaction with vessels containing pressurized liquids, 1994/1995). In this chapter, only the BAM experiment is used to evaluate the capability of FDS to predict BLEVE characteristics. By doing a little reminder on the BLEVE phenomenon, in 1998, the BAM conducted a BLEVE test with a road tank of 45 m3 of capacity, containing 5 tons of commercial propane (fill liquid level 22%) [19, 22]. The wagon was exposed to a fuel pool fire. In this test, an instrumentation has been performed to obtain physical quantities such as heat flux, temperature, and pressure. In the goal to make a comparison between empirical law and numerical modeling, the next sections will present the equations used for the empirical laws and the different models proposed to simulate the reactive flows inducted by the fireball. #### 2.3 BLEVE modeling using empirical laws In order to predict the fireball effects, different authors proposed correlations to predict fireball diameter and lifetime based on fuel quantity [4, 23–30]. These correlations are given in the following equations: $$D\_{\rm FB} = \mathfrak{a}\_1 \mathbf{M}^{b1} \tag{1}$$ $$\mathbf{t}\_{FB} = \mathfrak{a}\_2 \mathbf{M}^{b\_2} \tag{2}$$ where DFB is the fireball diameter, M is the fuel mass, tFB is the fireball lifetime, and a1, b1, a2, and b2 are empirical constants. With the difficulty to choose good coefficients which give better correlation for the fireball characterization, a comparative analysis made by Satyanarayana et al. [31] to define the best correlations which describe the fireball diameter and lifetime is given as follows: $$D\_{FB} = \mathsf{G.14} \, M^{0.325} \tag{3}$$ $$t\_{FB} = \mathbf{0}.41M^{0.340} \tag{4}$$ Equations (3) and (4) are used in this study in order to compare with the experiment data and CFD predictions. To estimate the incident radiation received by a target at a given distance, the solid-flame model may be used [23, 27]: $$ \dot{q}\_r^n = E\_p \cdot F\_v \cdot \pi\_{atm} \tag{5} $$ 125 section. written as [32]: where D<sup>∗</sup> the specific heat. δx computational cost [18, 39]. mends a D<sup>∗</sup> \_ BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… computing capability using message-passing interface (MPI) [26, 33]. The numerical modelings were performed using the CFD code FDS 6.5.3 [32]. This one solves the Navier–Stokes equations based on an explicit finite difference scheme. Moreover, it models the thermally driven flow with an emphasis on smoke and heat transport. It is a LES model using a uniform mesh and has parallel The modeling of the fire is based on a reaction rate considered as infinitely fast, and the combustion is modeled using the EDC of Magnussen and Hjertager [34–36]. The turbulent combustion processes are based on the governing equations for the mass fraction of the chemical species, such as Cx Hy, O2, CO2, H2O, and N<sup>2</sup> > y \_ 2 H2O + 3.76(x + y \_ 4 ) N<sup>2</sup> (6) , is ) (7) is the heat release rate, and cp is )(O<sup>2</sup> + 3.76 N2) → xCO<sup>2</sup> + Considering the complexity of the BLEVE phenomenon, only the fireball is modeled in this work. Indeed, as the published CFD studies say, the container disintegration is complicated to model and is not considered. For that, the present The fuel used is propane. Its heat of combustion is set to 46,334 kJ/kg. The ejection surface was calculated using the approach of Makhviladze et al. [38]. The fuel releases as a hot gas with a temperature equal to 700°C. The ignition of the mixture air/fuel is ensured by an autoignition. The extinction model and turbulence model The numerical simulations are carried out in a rectangular 3D domain with dimensions of 200 m × 200 m × 300 m assimilated to an open ambient environment. These dimensions are obtained from the max-diameter and the max-height of the fireball calculated using the empirical correlations presented in the second In the mesh resolution, it is necessary to determine the fire characteristic diameter according to its heat release rate (HRR). This diameter, denoted D<sup>∗</sup> = ( Q̇ \_ ρ∞ c∞ T∞ √ From obtaining the characteristic diameter, the optimal mesh size of the domain ratio between 4 and 16 to produce accurate results at a moderate Based on several experiences, the US Nuclear Regulatory Commission recom- In order to model a fireball using FDS, it is important to define the good mesh size. For that, a comparison between experiment data and numerical data using \_ g , where δx is the nominal mesh size. D∗ δx is the characteristic fire diameter, Q̇ DOI: http://dx.doi.org/10.5772/intechopen.92990 3. Numerical modeling of BLEVE 3.1 Fire source modeling Cx Hy + (x + 3.2 Mesh sensitivity analysis is given by the dimensionless ratio D<sup>∗</sup> \_ through a single step as follows [37]: y \_ 4 used in simulations are the default code models. study is based on the BLEVE modeling by fuel release. where q̇ <sup>r</sup> ″ is the radiation received by target,Ep is the surface emissive power, Fv is the view factor, and τatm is the atmospheric attenuation factor (transmissivity). BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 #### 3. Numerical modeling of BLEVE The numerical modelings were performed using the CFD code FDS 6.5.3 [32]. This one solves the Navier–Stokes equations based on an explicit finite difference scheme. Moreover, it models the thermally driven flow with an emphasis on smoke and heat transport. It is a LES model using a uniform mesh and has parallel computing capability using message-passing interface (MPI) [26, 33]. #### 3.1 Fire source modeling Fire Safety and Management Awareness pressurized liquids, 1994/1995). fireball. is given as follows: ment data and CFD predictions. solid-flame model may be used [23, 27]: FDS to predict BLEVE characteristics. 2.3 BLEVE modeling using empirical laws correlations are given in the following equations: and a1, b1, a2, and b2 are empirical constants. quantities such as heat flux, temperature, and pressure. instrumentation. However, the current measurement instruments do not allow the proper acquisition of results during a BLEVE test due to its magnitude. In addition, the high cost of this type of test and considering respect for the environment, there are few experimental tests that deal with this kind of phenomenon. In the literature [19, 22], there are large-scale experiment tests: the BAM test (Bundesanstalt für Materialforschung und –prüfung, Allemagne), the British Gas experiments, and the JIVE tests (hazards consequences of jet fire interaction with vessels containing In this chapter, only the BAM experiment is used to evaluate the capability of By doing a little reminder on the BLEVE phenomenon, in 1998, the BAM conducted a BLEVE test with a road tank of 45 m3 of capacity, containing 5 tons of commercial propane (fill liquid level 22%) [19, 22]. The wagon was exposed to a fuel pool fire. In this test, an instrumentation has been performed to obtain physical In the goal to make a comparison between empirical law and numerical modeling, the next sections will present the equations used for the empirical laws and the different models proposed to simulate the reactive flows inducted by the In order to predict the fireball effects, different authors proposed correlations to predict fireball diameter and lifetime based on fuel quantity [4, 23–30]. These where DFB is the fireball diameter, M is the fuel mass, tFB is the fireball lifetime, With the difficulty to choose good coefficients which give better correlation for the fireball characterization, a comparative analysis made by Satyanarayana et al. [31] to define the best correlations which describe the fireball diameter and lifetime Equations (3) and (4) are used in this study in order to compare with the experi- ″ is the radiation received by target,Ep is the surface emissive power, Fv is To estimate the incident radiation received by a target at a given distance, the the view factor, and τatm is the atmospheric attenuation factor (transmissivity). q̇ r n DFB = a1 M<sup>b</sup><sup>1</sup> (1) tFB = a2 M<sup>b</sup><sup>2</sup> (2) DFB = 6.14 M0.325 (3) tFB = 0.41 M0.340 (4) = Ep ⋅ Fv ⋅ τatm (5) 124 where q̇ r The modeling of the fire is based on a reaction rate considered as infinitely fast, and the combustion is modeled using the EDC of Magnussen and Hjertager [34–36]. The turbulent combustion processes are based on the governing equations for the mass fraction of the chemical species, such as Cx Hy, O2, CO2, H2O, and N<sup>2</sup> through a single step as follows [37]: $$\rm C\_xH\_y + \left(x + \frac{y}{4}\right)\left(O\_2 + 3.76N\_2\right) \rightarrow xCO\_2 + \frac{y}{2}H\_2O + 3.76\left(x + \frac{y}{4}\right)N\_2 \tag{6}$$ Considering the complexity of the BLEVE phenomenon, only the fireball is modeled in this work. Indeed, as the published CFD studies say, the container disintegration is complicated to model and is not considered. For that, the present study is based on the BLEVE modeling by fuel release. The fuel used is propane. Its heat of combustion is set to 46,334 kJ/kg. The ejection surface was calculated using the approach of Makhviladze et al. [38]. The fuel releases as a hot gas with a temperature equal to 700°C. The ignition of the mixture air/fuel is ensured by an autoignition. The extinction model and turbulence model used in simulations are the default code models. The numerical simulations are carried out in a rectangular 3D domain with dimensions of 200 m × 200 m × 300 m assimilated to an open ambient environment. These dimensions are obtained from the max-diameter and the max-height of the fireball calculated using the empirical correlations presented in the second section. #### 3.2 Mesh sensitivity analysis In the mesh resolution, it is necessary to determine the fire characteristic diameter according to its heat release rate (HRR). This diameter, denoted D<sup>∗</sup> , is written as [32]: = ( Q̇ \_ $$D^\* = \left(\frac{\dot{Q}}{\rho\_{\text{ov}} c\_{\text{ov}} T\_{\text{ov}} \sqrt{\xi}}\right) \tag{7}$$ where D<sup>∗</sup> is the characteristic fire diameter, Q̇ is the heat release rate, and cp is the specific heat. From obtaining the characteristic diameter, the optimal mesh size of the domain is given by the dimensionless ratio D<sup>∗</sup> \_ δx , where δx is the nominal mesh size. Based on several experiences, the US Nuclear Regulatory Commission recommends a D<sup>∗</sup> \_ δx ratio between 4 and 16 to produce accurate results at a moderate computational cost [18, 39]. In order to model a fireball using FDS, it is important to define the good mesh size. For that, a comparison between experiment data and numerical data using four mesh sizes is made in Figure 1(a) and (b). The different mesh sizes are obtained from the US Nuclear Regulatory Commission recommendation. The numerical simulations are carried out in a rectangular 3D domain with dimensions of 200 m × 200 m × 300 m as mentioned previously. The comparisons between the experiment and the predictions for the four different meshes are made based on the evolution of the heat flux and the fireball height (cf. Figure 1). The heat flux was measured at 30 m over the ground from the projected center of the fireball on the ground under the fireball, and the height was obtained from the fireball center to the ground level. These figures show that the numerical results obtained from the mesh sizes of 0.5 m and 1 m converge with the experimental results, while the results from the mesh sizes of 2 m and 4 m diverge. Moreover, the mesh size of 0.5 m offers more precision than the results obtained with a mesh size of 1 m as shown by the root-mean-square Error (cf. Table 1). From Figure 1(a) and (b), the numerical simulation with a mesh size of 0.5 m is more precise but requires a calculation time 50 times greater than the calculation carried out with a mesh size of 1 m (cf. Table 1). Thus, by wanting to conciliate precision and optimal calculation time, the mesh size of 1 m will be used for the rest of numerical simulations. This mesh size allows solving the Navier–Stokes equations with a good accuracy. Indeed, with the mesh size of 1 m, the different numerical models such as the turbulence model based on the Deardorff model, the combustion model based on the EDC definition, and the extinction model based on the critical temperature flame are very well calculated for giving a very nice modeling of the fireball. Moreover, taking into account the mesh size Figure 1. Mesh resolution on (a) the height of fireball center and (b) the heat flux at 30 m on ground level. 127 Figure 2. and (d) 6 s. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… Simulation of the fireball temperature field with mesh size 1 m in the cross-section at (a) 2 s, (b) 3 s, (c) 4 s, DOI: http://dx.doi.org/10.5772/intechopen.92990 #### Table 1. Results of mesh sensitivity analysis. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 Figure 2. Simulation of the fireball temperature field with mesh size 1 m in the cross-section at (a) 2 s, (b) 3 s, (c) 4 s, and (d) 6 s. Fire Safety and Management Awareness Error (cf. Table 1). of 200 m × 200 m × 300 m as mentioned previously. four mesh sizes is made in Figure 1(a) and (b). The different mesh sizes are obtained from the US Nuclear Regulatory Commission recommendation. The numerical simulations are carried out in a rectangular 3D domain with dimensions The comparisons between the experiment and the predictions for the four different meshes are made based on the evolution of the heat flux and the fireball height (cf. Figure 1). The heat flux was measured at 30 m over the ground from the projected center of the fireball on the ground under the fireball, and the height was obtained from the fireball center to the ground level. These figures show that the numerical results obtained from the mesh sizes of 0.5 m and 1 m converge with the experimental results, while the results from the mesh sizes of 2 m and 4 m diverge. Moreover, the mesh size of 0.5 m offers more precision than the results obtained with a mesh size of 1 m as shown by the root-mean-square From Figure 1(a) and (b), the numerical simulation with a mesh size of 0.5 m is more precise but requires a calculation time 50 times greater than the calculation carried out with a mesh size of 1 m (cf. Table 1). Thus, by wanting to conciliate precision and optimal calculation time, the mesh size of 1 m will be used for the rest of numerical simulations. This mesh size allows solving the Navier–Stokes equations with a good accuracy. Indeed, with the mesh size of 1 m, the different numerical models such as the turbulence model based on the Deardorff model, the combustion model based on the EDC definition, and the extinction model based on the critical temperature flame are very well calculated for giving a very nice modeling of the fireball. Moreover, taking into account the mesh size Numerical grid Number of cells Root-mean-square error CPU time (min) Mesh size 4 m 187,500 60.22 34.09 2 Mesh size 2 m 1,500,000 59.01 21.56 14 Mesh size 1 m 12,000,000 9.74 16.24 161 Mesh size 0.5 m 96,000,000 5.86 13.23 8000 Mesh resolution on (a) the height of fireball center and (b) the heat flux at 30 m on ground level. Height (m) Heat flux (kW/m2 ) 126 Table 1. Figure 1. Results of mesh sensitivity analysis. Table 2. Comparison between numerical data and BAM test. of 2 and 4 m, there is an important divergency on the solving of the previous numerical models. Working with the mesh size of 1 m, Figure 2(a)–(d) shows the evolution and the development of the fireball structure at different times (2, 3, 4, and 6 s) after the fuel release to the atmosphere. From these pictures, the evolutions of the temperature field obtained from the numerical modeling highlight the same observations made by Hurley et al. [40]. It is observed that the diameter of the flame increases the height and the time, and Hurley et al. have observed that the diameter of the fireball reaches its maximum at about 6 s with a value of 100 m as diameter. And, by making a comparison with the numerical data, this one agrees with experimental results. Moreover, considering that the flame temperature of a hydrocarbon fire can approach about 1300°C, it is shown in Figure 2(a)–(d) that the predicted field temperature represents the diameter of the fireball during its evolution. In this context, the reactive flows modeled using this mesh resolution come close themselves to the flame dynamics of BLEVE phenomenon. In conclusion, FDS can predict BLEVE characteristics after a good definition of the mesh size and the fuel release rate. For another case of validation, Table 2 illustrates the comparison between numerical data and BAM test. In this one, it is observed that the predictions of the parameters such as max-diameter, lifetime, and max-height of the fireball agree with experiment with a better precision than empirical estimates. #### 4. BLEVE thermal effects: case study for a Hassi R'Mel gas processing plant From the previous analyses, it has been shown that FDS code is able to simulate the evolution and development of a fireball in comparison with experimental test, considering that it is possible to predict the evolution and thermal effects of a BLEVE in a real installation. In addition, from the numerical results obtained in the previous section, it is necessary to use a nice mesh size in order to make an accurate modeling of a fireball under FDS and a good knowledge of the mass and the release rate of the fuel. Moreover, the definition of a calculation domain that considers the recirculation and the reactive flows during the fireball expansion is very important to justify a good numerical calculation. So, respecting the previous numerical recommendations, it is possible to simulate thermal effects of BLEVE in a real installation such as in Hassi R'Mel Gas Processing Plant. #### 4.1 Description of the gas processing plant and the ignition source The gas processing plant studied in this work is defined as the Module Processing Plant 3 (MPP3) of SONATRACH Company at Hassi R'Mel gas field 129 Table 3. Figure 3. Numerical MPP3-plant. Volume (m3 Propane density (kg/m3 Technical characteristics of the accumulator D108. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… (located about 550 km south of Algiers). This MPP3-plant consists of three identical gas processing trains that mainly produce natural gas (with a produc- the configuration of the MPP3-plant. The origin of the explosion is taken at the level of a pressurized propane accumulator D108 located in the MPP3-plant as The choice of the accumulator D108 is based on the opinions of the risk analysts who consider it as one of the most critical systems in the MPP3-plant, which can generate catastrophic BLEVE accidents [41]. Table 3 summarizes the technical The numerical modeling of the MPP3-plant described above is carried out in an open calculation domain of 300 m × 300 m × 360 m. The dimensions of this domain are chosen based on the fireball diameter and height calculated using empirical correlations. The calculations are carried out under atmospheric conditions with a relative humidity of 40% and an ambient temperature of 20°C. The plant configuration is modeled as solid obstructions considering the real equipment dimensions The calculations were performed with a time step of 0.01 s and took 2729 minutes with a mesh size of 1 m (i.e., 32,400,000 meshes) using 90 CPUs. The simulation is performed using the default numerical models. The ejection surface was calculated using the approach of Makhviladze et al. [38] as mentioned in Section 3. The origin of the explosion is taken at the level of the D108 as mentioned previously. Using the Characteristics Values Operating temperature (°C) 40 Operating pressure (bar) 14.5 ) 50 ) 483.6 /day), LPG, and condensate. Figure 3 illustrates DOI: http://dx.doi.org/10.5772/intechopen.92990 characteristics of the D108 vessel used in our calculation. tion capacity of 60 million m3 shown in Figure 3. 4.2 Boundary conditions of the three MPP3-plant trains. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 (located about 550 km south of Algiers). This MPP3-plant consists of three identical gas processing trains that mainly produce natural gas (with a production capacity of 60 million m3 /day), LPG, and condensate. Figure 3 illustrates the configuration of the MPP3-plant. The origin of the explosion is taken at the level of a pressurized propane accumulator D108 located in the MPP3-plant as shown in Figure 3. The choice of the accumulator D108 is based on the opinions of the risk analysts who consider it as one of the most critical systems in the MPP3-plant, which can generate catastrophic BLEVE accidents [41]. Table 3 summarizes the technical characteristics of the D108 vessel used in our calculation. #### 4.2 Boundary conditions Fire Safety and Management Awareness Comparison between numerical data and BAM test. numerical models. Fireball Characteristics Table 2. with experimental results. empirical estimates. processing plant flame dynamics of BLEVE phenomenon. of 2 and 4 m, there is an important divergency on the solving of the previous Experiment Empirical Present Max-diameter (m) 100 98 101 1.41 0.71 Duration (s) 7.2 7.4 7.8 0.14 0.42 Max-height (m) 100 74 99 18.38 0.71 data RMSE (Empirical) RMSE (Present data) Working with the mesh size of 1 m, Figure 2(a)–(d) shows the evolution and the development of the fireball structure at different times (2, 3, 4, and 6 s) after the fuel release to the atmosphere. From these pictures, the evolutions of the temperature field obtained from the numerical modeling highlight the same observations made by Hurley et al. [40]. It is observed that the diameter of the flame increases the height and the time, and Hurley et al. have observed that the diameter of the fireball reaches its maximum at about 6 s with a value of 100 m as diameter. And, by making a comparison with the numerical data, this one agrees Moreover, considering that the flame temperature of a hydrocarbon fire can approach about 1300°C, it is shown in Figure 2(a)–(d) that the predicted field temperature represents the diameter of the fireball during its evolution. In this context, the reactive flows modeled using this mesh resolution come close themselves to the In conclusion, FDS can predict BLEVE characteristics after a good definition of the mesh size and the fuel release rate. For another case of validation, Table 2 illustrates the comparison between numerical data and BAM test. In this one, it is observed that the predictions of the parameters such as max-diameter, lifetime, and max-height of the fireball agree with experiment with a better precision than From the previous analyses, it has been shown that FDS code is able to simulate the evolution and development of a fireball in comparison with experimental test, considering that it is possible to predict the evolution and thermal effects of a BLEVE in a real installation. In addition, from the numerical results obtained in the previous section, it is necessary to use a nice mesh size in order to make an accurate modeling of a fireball under FDS and a good knowledge of the mass and the release rate of the fuel. Moreover, the definition of a calculation domain that considers the recirculation and the reactive flows during the fireball expansion is very important to justify a good numerical calculation. So, respecting the previous numerical recommendations, it is possible to simulate thermal effects of BLEVE in 4. BLEVE thermal effects: case study for a Hassi R'Mel gas a real installation such as in Hassi R'Mel Gas Processing Plant. 4.1 Description of the gas processing plant and the ignition source The gas processing plant studied in this work is defined as the Module Processing Plant 3 (MPP3) of SONATRACH Company at Hassi R'Mel gas field 128 The numerical modeling of the MPP3-plant described above is carried out in an open calculation domain of 300 m × 300 m × 360 m. The dimensions of this domain are chosen based on the fireball diameter and height calculated using empirical correlations. The calculations are carried out under atmospheric conditions with a relative humidity of 40% and an ambient temperature of 20°C. The plant configuration is modeled as solid obstructions considering the real equipment dimensions of the three MPP3-plant trains. The calculations were performed with a time step of 0.01 s and took 2729 minutes with a mesh size of 1 m (i.e., 32,400,000 meshes) using 90 CPUs. The simulation is performed using the default numerical models. The ejection surface was calculated using the approach of Makhviladze et al. [38] as mentioned in Section 3. The origin of the explosion is taken at the level of the D108 as mentioned previously. Using the Figure 3. Numerical MPP3-plant. #### Table 3. Technical characteristics of the accumulator D108. same modeling approach presented in Section 3, the BLEVE is modeled through the ejection of 24,180 kg of hot propane with a velocity of 100 m/s. #### 5. Results and discussions In the previous section, it is shown that the comparison of the predicted fireball diameter and lifetime with the empirical values is similar to the experimental data. However, the predicted height is better than the empirical value in comparison with experiment data. Considering the real installation, there are no experimental data and so no possibility to compare with empirical values and numerical data. In these conditions, the comparison is made only between the numerical and empirical data based on the evaluation of BLEVE characteristics. Moreover, considering the observations made in the previous section, the results issued from the BLEVE simulation in the MPP3-plant show similar observations. Indeed, in Table 4, the predicted fireball diameter and lifetime are like the empirical values, but the empirical height is underestimated by comparing to the predicted value. Taking into account the comparisons obtained previously, it is possible to say that the evolution and the development of the fireball predicted by FDS in the MPP3-plant would be representative of reality. Figure 4 shows the simulation of the fireball at two different times in the studied plant. With this simulation, it is possible to follow the evolution of different physical parameters in a spatiotemporal manner such as heat flux, heat release rate, species concentrations, flame temperature, etc. In this paper, only the prediction of heat flux is studied. Figure 5 presents the comparison between the prediction and the empirical approach based on the evolution of heat flux over time at 50 and 70 m at ground level. It is found that the prediction provides a temporal evolution of the heat flux representative of the reality in comparison with the empirical one which gives a constant value. Indeed, during the first moments, a maximum peak of the heat flux is observed. This maximum value represents the heat flux emitted by the fireball when the latter is near to the ground. With the fireball elevation in height, the heat flux received at ground level decreases. This is represented by the evolution of the heat flux predicted by FDS code. From these comparisons, it is justified that the data provided by the numerical simulation give a more realistic support during a QRA. Indeed, as indicated in introduction, risk analysis requires knowledge of representative input data of the phenomenon to be studied. Thus, depending on the data, a risk analysis can be well estimated, underestimated, and overestimated. As a result, it is preferable to use the data obtained from numerical simulation in comparison with the data obtained from empirical laws. 131 Figure 4. Figure 5. ground level. Fireball simulation at (a) 2 s and (b) 8 s. height less than 70 m. also a tool that can be used in a QRA. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… In addition to the evolutions of the heat flux presented in Figure 5, the same observation is found in Figure 6(a) and (b). These figures show the heat flux distribution at 1 s and 4 s in order to better observe the heat flux field over the entire MPP3-plant. With this illustration, it is shown that it is necessary to present the results during the first few seconds. Indeed, considering the heat flux distribution throughout the plant, it is observed during the first instants that the heat flux intensity is important at the explosion source and decreases in the remote zones. In Figure 6, it is observed that the heat flux intensity decreases at the explosion Comparison between empirical and thermal flux prediction at the distance of (a) 50 m and (b) 75 m on the origin and increases in the remote zones with the fireball evolution in terms of diameter and height. This observation is like the reality and is true only for a fireball In conclusion, the BLEVE thermal effects in Hassi R'Mel Gas Processing Plant are well predicted by FDS. In addition, the predictions of FDS give information which allows a better understanding on BLEVE phenomenon. It can be considered DOI: http://dx.doi.org/10.5772/intechopen.92990 Table 4. Comparison between numerical and empirical data for MPP3-plant. BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 Figure 4. Fireball simulation at (a) 2 s and (b) 8 s. Figure 5. Fire Safety and Management Awareness 5. Results and discussions experiment data. studied. ing a QRA. same modeling approach presented in Section 3, the BLEVE is modeled through the In the previous section, it is shown that the comparison of the predicted fireball diameter and lifetime with the empirical values is similar to the experimental data. However, the predicted height is better than the empirical value in comparison with Considering the real installation, there are no experimental data and so no possibility to compare with empirical values and numerical data. In these conditions, the comparison is made only between the numerical and empirical data based on the evaluation of BLEVE characteristics. Moreover, considering the observations made in the previous section, the results issued from the BLEVE simulation in the MPP3-plant show similar observations. Indeed, in Table 4, the predicted fireball diameter and lifetime are like the empirical values, but the empirical height is Taking into account the comparisons obtained previously, it is possible to say that the evolution and the development of the fireball predicted by FDS in the MPP3-plant would be representative of reality. Figure 4 shows the simulation of the fireball at two different times in the studied plant. With this simulation, it is possible to follow the evolution of different physical parameters in a spatiotemporal manner such as heat flux, heat release rate, species concentrations, flame temperature, etc. In this paper, only the prediction of heat flux is Figure 5 presents the comparison between the prediction and the empirical approach based on the evolution of heat flux over time at 50 and 70 m at ground level. It is found that the prediction provides a temporal evolution of the heat flux representative of the reality in comparison with the empirical one which gives a constant value. Indeed, during the first moments, a maximum peak of the heat flux is observed. This maximum value represents the heat flux emitted by the fireball when the latter is near to the ground. With the fireball elevation in height, the heat flux received at ground level decreases. This is represented by the evolution of the heat flux predicted by FDS code. From these comparisons, it is justified that the data provided by the numerical simulation give a more realistic support dur- Indeed, as indicated in introduction, risk analysis requires knowledge of representative input data of the phenomenon to be studied. Thus, depending on the data, a risk analysis can be well estimated, underestimated, and overestimated. As a result, it is preferable to use the data obtained from numerical simulation in com- Fireball characteristics Empirical Present data Max-diameter (m) 163 174 Duration (s) 12.7 14 Max-height (m) 122 160 ejection of 24,180 kg of hot propane with a velocity of 100 m/s. underestimated by comparing to the predicted value. parison with the data obtained from empirical laws. Comparison between numerical and empirical data for MPP3-plant. 130 Table 4. Comparison between empirical and thermal flux prediction at the distance of (a) 50 m and (b) 75 m on the ground level. In addition to the evolutions of the heat flux presented in Figure 5, the same observation is found in Figure 6(a) and (b). These figures show the heat flux distribution at 1 s and 4 s in order to better observe the heat flux field over the entire MPP3-plant. With this illustration, it is shown that it is necessary to present the results during the first few seconds. Indeed, considering the heat flux distribution throughout the plant, it is observed during the first instants that the heat flux intensity is important at the explosion source and decreases in the remote zones. In Figure 6, it is observed that the heat flux intensity decreases at the explosion origin and increases in the remote zones with the fireball evolution in terms of diameter and height. This observation is like the reality and is true only for a fireball height less than 70 m. In conclusion, the BLEVE thermal effects in Hassi R'Mel Gas Processing Plant are well predicted by FDS. In addition, the predictions of FDS give information which allows a better understanding on BLEVE phenomenon. It can be considered also a tool that can be used in a QRA. Figure 6. Thermal radiation contour plot in the x-y plane at (a) 1 s and (b) 4 s. ## 6. Conclusion In this chapter, a CFD evaluation of the thermal effects of the BLEVE phenomenon in a real installation is presented. This evaluation firstly required the code validation to correctly simulate the BLEVE characteristics in comparison with the data that come from literature experimental test. Numerical calculations were performed using the CFD FDS code version 6.5.3 with the default numerical models. The results show a good agreement between the predictions and the experiments, justifying a nice capability to FDS to simulate the fireball dynamics with a good accuracy. After highlighting that FDS can predict the spatiotemporal evolution of a fireball in comparison with an experimental test, a simulation of the BLEVE is performed in a real installation. This involves studying the fireball thermal effects resulting from the explosion of a pressurized propane tank in an Algerian gas treatment unit. The results obtained showed great relevance of carrying out this type of study in 133 Author details Brady Manescau1 Fatiha Zidani<sup>2</sup> \, Khaled Chetehouna1 2 – Mostefa Ben Boulaïd, Fesdis, Batna, Algeria provided the original work is properly cited. Kasdi Merbah University – Ouargla, Ouargla, Algeria \Address all correspondence to: [email protected] 1 INSA Centre Val de Loire, Univ. Orléans, PRISME, Bourges, France 2 LRPI Laboratory, Institute of Health and Industrial Safety, University of Batna 3 DIRE Laboratory, Department of Applied Engineering, Institute of Technology, © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, , Ilyas Sellami1,2,3, Rachid Nait-Said3 and BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… this type of installation. From the numerical data, it is shown that the heat flux reaches a maximum value during the first moments at ground level and decreases with the elevation of the fireball. In addition, comparisons between prediction and empirical models, based on heat flux evolution, show that prediction is representative of reality compared to empirical models. Thus, for a risk analysis in this type of Moreover, the current results can be considered as a first step to make a modeling of the BLEVE phenomenon, and in order to improve the global description of this phenomenon, it will be necessary to consider, in a next work, the container DOI: http://dx.doi.org/10.5772/intechopen.92990 installation, it is preferable to use the numerical approach. disintegration in order to model the complete BLEVE process. #### BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 this type of installation. From the numerical data, it is shown that the heat flux reaches a maximum value during the first moments at ground level and decreases with the elevation of the fireball. In addition, comparisons between prediction and empirical models, based on heat flux evolution, show that prediction is representative of reality compared to empirical models. Thus, for a risk analysis in this type of installation, it is preferable to use the numerical approach. Moreover, the current results can be considered as a first step to make a modeling of the BLEVE phenomenon, and in order to improve the global description of this phenomenon, it will be necessary to consider, in a next work, the container disintegration in order to model the complete BLEVE process. ## Author details Fire Safety and Management Awareness 132 6. Conclusion Figure 6. In this chapter, a CFD evaluation of the thermal effects of the BLEVE phenomenon in a real installation is presented. This evaluation firstly required the code validation to correctly simulate the BLEVE characteristics in comparison with the data that come from literature experimental test. Numerical calculations were performed using the CFD FDS code version 6.5.3 with the default numerical models. The results show a good agreement between the predictions and the experiments, justifying a nice capability to FDS to simulate the fireball dynamics with a good accuracy. Thermal radiation contour plot in the x-y plane at (a) 1 s and (b) 4 s. After highlighting that FDS can predict the spatiotemporal evolution of a fireball in comparison with an experimental test, a simulation of the BLEVE is performed in a real installation. This involves studying the fireball thermal effects resulting from the explosion of a pressurized propane tank in an Algerian gas treatment unit. The results obtained showed great relevance of carrying out this type of study in Brady Manescau1 \, Khaled Chetehouna1 , Ilyas Sellami1,2,3, Rachid Nait-Said3 and Fatiha Zidani<sup>2</sup> 1 INSA Centre Val de Loire, Univ. Orléans, PRISME, Bourges, France 2 LRPI Laboratory, Institute of Health and Industrial Safety, University of Batna 2 – Mostefa Ben Boulaïd, Fesdis, Batna, Algeria 3 DIRE Laboratory, Department of Applied Engineering, Institute of Technology, Kasdi Merbah University – Ouargla, Ouargla, Algeria \Address all correspondence to: [email protected] © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. #### References [1] Sellami I, Nait-Said R. Analyse Quantitative des Risques Industriels: Apport des Techniques Floues et Possibilistes. Saarbrücken: Éditions Universitaires EuropÉennes; 2017 [2] CCPS. Guidelines for Vapor Cloud Explosion, Pressure Vessel Burst, BLEVE, and Flash Fire Hazards. 2nd ed. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2010 [3] Abbasi T, Abbasi SA. The boiling liquid expanding vapour explosion (BLEVE): Mechanism, consequence assessment, management. Journal of Hazardous Materials. 2007;141(3):489-519 [4] Roberts AF. Thermal radiation hazards from releases of LPG from pressurised storage. Fire Safety Journal. 1981;4(3):197-212 [5] Crocker WP, Napier DH. Assessment of mathematical models for fire and explosion hazards of liquefied petroleum gases. Journal of Hazardous Materials. 1988;20(C):109-135 [6] Prugh RW. Quantitative evaluation of 'Bleve' hazards. Journal of Fire Protection Engineering. 1991;3(1):9-24 [7] Roberts T, Gosse A, Hawksworth S. Thermal radiation from fireballs on failure of liquefied petroleum gas storage vessels. Process Safety and Environment Protection. 2000;78(3):184-192 [8] Birk AM, Davison C, Cunningham M. Blast overpressures from medium scale BLEVE tests. Journal of Loss Prevention in the Process Industries. 2007;20(3):194-206 [9] Bubbico R, Marchini M. Assessment of an explosive LPG release accident: A case study. Journal of Hazardous Materials. 2008;155(3):558-565 [10] Chen S, Sun J, Wan W. Boiling liquid expanding vapor explosion: Experimental research in the evolution of the two-phase flow and over-pressure. Journal of Hazardous Materials. 2008;156(1-3):530-537 [11] Laboureur D, Heymes F, Lapebie E, Buchlin JM, Rambaud P. BLEVE overpressure: Multiscale comparison of blast wave modeling. Process Safety Progress. 2014;33(3):274-284 [12] Laboureur D et al. A closer look at BLEVE overpressure. Process Safety and Environment Protection. May 2015;95:159-171 [13] Van Den Berg AC, Van Der Voort MM, Weerheijm J, Versloot NHA. Expansion-controlled evaporation: A safe approach to BLEVE blast. Journal of Loss Prevention in the Process Industries. 2004;17(6):397-405 [14] Yakush SE, Makhviladze GM. Large Eddy simulation of hydrocarbon fireballs Institute for Problems in mechanics. In: Proceedings of the European Combustion Meeting. 2005 [15] Roper F, Arno J, Jaggers HC. The effect of release velocity and geometry on burning times for non-premixed fuel gas clouds. Combustion Science and Technology. 1991;78(4-6):315-338 [16] Luther W, Müller WC. FDS simulation of the fuel fireball from a hypothetical commercial airliner crash on a generic nuclear power plant. Nuclear Engineering and Design. 2009;239(10):2056-2069 [17] Rajendram A, Khan F, Garaniya V. Modelling of fire risks in an offshore facility. Fire Safety Journal. 2015;71:79-85 [18] Sikanen T, Hostikka S. Numerical simulations of liquid spreading and 135 BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… [28] Papazoglou IA, Aneziris ON. Uncertainty quantification in the health consequences of the boiling liquid expanding vapour explosion phenomenon. Journal of Hazardous Materials. 1999;67(3):217-235 [29] TNO. Methods for the Calculation of Physical Effects (Yellow Book): CPR 14E. 3rd ed. The Hague: Committee for the Prevention of Disasters; 2005 [30] TNO. Guidelines for Quantitative Risk Assessment (Purple Book): CPR 18E. Den Haag: Sdu Uitgevers; 1999 [31] Satyanarayana K, Borah M, Rao PG. Prediction of thermal hazards from fireballs. Journal of Loss Prevention in the Process Industries. [32] McGrattan K, Hostikka S, [34] Magnussen BF. The Eddy Combustion. 2005. pp. 21-24 1977;16(1):719-729 Edwards; 2005 McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator, [33] Gropp W, Lusk E, Skjellum A. Using MPI: Portable Parallel Programming with the Message-Passing Interface. dissipation concept- a bridge between science and technology. In: ECCOMAS Thematic Conference on Computational [35] Magnussen BF, Hjertager BH. On mathematical modeling of turbulent combustion with special emphasis on soot formation and combustion. Symposium on Combustion. [36] Poinsot T, Veynante D. Theoretical and Numerical Combustion. 2nd ed. McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator Technical Reference Guide Volume 1: [37] McGrattan K, Hostikka S, Mathematical Model. 2017 1991;4(5):344-347 User's Guide. 2015 2000 DOI: http://dx.doi.org/10.5772/intechopen.92990 [19] Balke C, Heller W, Konersmann R, Ludwig J. Study of the Failure Limits of a Railway Tank Car Filled with Liquefied Petroleum Gas Subjected to an Open Poolfire Test. Berlin: Germany; fires following an aircraft impact. Nuclear Engineering and Design. [20] Mishra KB, Wehrstedt KD, Krebs H. Boiling liquid expanding vapour explosion (BLEVE) of Peroxyfuels: Experiments and computational fluid dynamics (CFD) simulation. Physics Procedia. 2015;66(1):149-152 [21] Shelke A, Maheshwari NK, Gera B, Singh RK. CFD analysis of hydrocarbon fireballs. Combustion Science and Technology. 2017;189(8):1440-1466 [22] INERIS. OMEGA 5- Le BLEVE: phénoménologie et modélisation des effets thermiques. Institut National de l'Environnement Industriel et des [23] Casal J. Evaluation of the Effects and Consequences of Major Accidents in Industrial Plants. Vol. 8. Amsterdam: [24] CCPS. Guidelines for Chemical Process Quantitative Risk Analysis. 2nd ed. New York: American Institute of [25] CCPS. Guidelines for Consequence Cagin T. Multiscale Modeling for Process Safety Applications. Butterworth- [27] Mannan S. Lee's Loss Prevention in the Process Industries: Hazard Identification, Assessment, and Control. 4th ed. Amsterdam: Butterworth- Chemical Engineers; 2000 Analysis of Chemical Releases. New York: American Institute of Chemical Engineers; 1999 [26] Chakrabarty A, Mannan S, Heinemann; 2016 Heinemann Elsevier; 2012 Risques; 2002 Elsevier; 2008 2017;318:147-162 1999 BLEVE Fireball Effects in a Gas Industry: A Numerical Modeling Applied to the Case… DOI: http://dx.doi.org/10.5772/intechopen.92990 fires following an aircraft impact. Nuclear Engineering and Design. 2017;318:147-162 [19] Balke C, Heller W, Konersmann R, Ludwig J. Study of the Failure Limits of a Railway Tank Car Filled with Liquefied Petroleum Gas Subjected to an Open Poolfire Test. Berlin: Germany; 1999 [20] Mishra KB, Wehrstedt KD, Krebs H. Boiling liquid expanding vapour explosion (BLEVE) of Peroxyfuels: Experiments and computational fluid dynamics (CFD) simulation. Physics Procedia. 2015;66(1):149-152 [21] Shelke A, Maheshwari NK, Gera B, Singh RK. CFD analysis of hydrocarbon fireballs. Combustion Science and Technology. 2017;189(8):1440-1466 [22] INERIS. OMEGA 5- Le BLEVE: phénoménologie et modélisation des effets thermiques. Institut National de l'Environnement Industriel et des Risques; 2002 [23] Casal J. Evaluation of the Effects and Consequences of Major Accidents in Industrial Plants. Vol. 8. Amsterdam: Elsevier; 2008 [24] CCPS. Guidelines for Chemical Process Quantitative Risk Analysis. 2nd ed. New York: American Institute of Chemical Engineers; 2000 [25] CCPS. Guidelines for Consequence Analysis of Chemical Releases. New York: American Institute of Chemical Engineers; 1999 [26] Chakrabarty A, Mannan S, Cagin T. Multiscale Modeling for Process Safety Applications. Butterworth-Heinemann; 2016 [27] Mannan S. Lee's Loss Prevention in the Process Industries: Hazard Identification, Assessment, and Control. 4th ed. Amsterdam: Butterworth-Heinemann Elsevier; 2012 [28] Papazoglou IA, Aneziris ON. Uncertainty quantification in the health consequences of the boiling liquid expanding vapour explosion phenomenon. Journal of Hazardous Materials. 1999;67(3):217-235 [29] TNO. Methods for the Calculation of Physical Effects (Yellow Book): CPR 14E. 3rd ed. The Hague: Committee for the Prevention of Disasters; 2005 [30] TNO. Guidelines for Quantitative Risk Assessment (Purple Book): CPR 18E. Den Haag: Sdu Uitgevers; 1999 [31] Satyanarayana K, Borah M, Rao PG. Prediction of thermal hazards from fireballs. Journal of Loss Prevention in the Process Industries. 1991;4(5):344-347 [32] McGrattan K, Hostikka S, McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator, User's Guide. 2015 [33] Gropp W, Lusk E, Skjellum A. Using MPI: Portable Parallel Programming with the Message-Passing Interface. 2000 [34] Magnussen BF. The Eddy dissipation concept- a bridge between science and technology. In: ECCOMAS Thematic Conference on Computational Combustion. 2005. pp. 21-24 [35] Magnussen BF, Hjertager BH. On mathematical modeling of turbulent combustion with special emphasis on soot formation and combustion. Symposium on Combustion. 1977;16(1):719-729 [36] Poinsot T, Veynante D. Theoretical and Numerical Combustion. 2nd ed. Edwards; 2005 [37] McGrattan K, Hostikka S, McDermott R, Floyd J, Weinschenk C, Overholt K. Fire Dynamics Simulator Technical Reference Guide Volume 1: Mathematical Model. 2017 134 Fire Safety and Management Awareness [1] Sellami I, Nait-Said R. Analyse Quantitative des Risques Industriels: Apport des Techniques Floues et Possibilistes. Saarbrücken: Éditions Universitaires EuropÉennes; 2017 [10] Chen S, Sun J, Wan W. Boiling liquid expanding vapor explosion: Experimental research in the evolution of the two-phase flow and over-pressure. Journal of Hazardous Materials. 2008;156(1-3):530-537 [11] Laboureur D, Heymes F, Lapebie E, Buchlin JM, Rambaud P. 2014;33(3):274-284 2015;95:159-171 [13] Van Den Berg AC, Van Der Voort MM, Weerheijm J, BLEVE overpressure: Multiscale comparison of blast wave modeling. Process Safety Progress. [12] Laboureur D et al. A closer look at BLEVE overpressure. Process Safety and Environment Protection. May Versloot NHA. Expansion-controlled evaporation: A safe approach to BLEVE blast. Journal of Loss Prevention in the Process Industries. 2004;17(6):397-405 [14] Yakush SE, Makhviladze GM. Large Eddy simulation of hydrocarbon fireballs Institute for Problems in mechanics. In: Proceedings of the European Combustion Meeting. 2005 [15] Roper F, Arno J, Jaggers HC. The effect of release velocity and geometry on burning times for non-premixed fuel gas clouds. Combustion Science and Technology. 1991;78(4-6):315-338 [16] Luther W, Müller WC. FDS simulation of the fuel fireball from a hypothetical commercial airliner crash on a generic nuclear power plant. Nuclear Engineering and Design. [17] Rajendram A, Khan F, Garaniya V. offshore facility. Fire Safety Journal. [18] Sikanen T, Hostikka S. Numerical simulations of liquid spreading and 2009;239(10):2056-2069 Modelling of fire risks in an 2015;71:79-85 [2] CCPS. Guidelines for Vapor Cloud Explosion, Pressure Vessel Burst, BLEVE, and Flash Fire Hazards. 2nd ed. Hoboken, NJ, USA: John Wiley & Sons, [3] Abbasi T, Abbasi SA. The boiling liquid expanding vapour explosion (BLEVE): Mechanism, consequence assessment, management. Journal of Hazardous Materials. [4] Roberts AF. Thermal radiation hazards from releases of LPG from pressurised storage. Fire Safety Journal. of mathematical models for fire and explosion hazards of liquefied petroleum gases. Journal of Hazardous Materials. 1988;20(C):109-135 [5] Crocker WP, Napier DH. Assessment [6] Prugh RW. Quantitative evaluation of 'Bleve' hazards. Journal of Fire Protection Engineering. 1991;3(1):9-24 [7] Roberts T, Gosse A, Hawksworth S. Thermal radiation from fireballs on failure of liquefied petroleum gas storage vessels. Process Safety and Environment Protection. Cunningham M. Blast overpressures from medium scale BLEVE tests. Journal [9] Bubbico R, Marchini M. Assessment of an explosive LPG release accident: A case study. Journal of Hazardous Materials. 2008;155(3):558-565 of Loss Prevention in the Process Industries. 2007;20(3):194-206 2007;141(3):489-519 1981;4(3):197-212 2000;78(3):184-192 [8] Birk AM, Davison C, Inc.; 2010 References [38] Makhviladze GM, Roberts JP, Yakush SE. Formation and combustion of gas clouds in accidental discharge to the atmosphere. Combustion, Explosion, and Shock Waves. 1997;33(2):144-156 [39] Magnognou B, Garo JP, Coudour B, Wang HY. Risk analysis of unburnt gas ignition in an exhaust system connected to a confined and mechanically ventilated enclosure fire. Fire Safety Journal. July 2017;91:291-302 [40] Hurley MJ et al. SFPE Handbook of Fire Protection Engineering. Fifth ed. 2016 [41] SONATRACH. Risk Assessment Study of Industrial Units, Report N° EP002715. Hassi R'Mel; 2010 Fire Safety and Management Awareness [38] Makhviladze GM, Roberts JP, Yakush SE. Formation and combustion of gas clouds in accidental discharge to the atmosphere. Combustion, Explosion, and Shock Waves. [39] Magnognou B, Garo JP, Coudour B, Wang HY. Risk analysis of unburnt gas ignition in an exhaust system connected [40] Hurley MJ et al. SFPE Handbook of Fire Protection Engineering. Fifth ed. [41] SONATRACH. Risk Assessment Study of Industrial Units, Report N° to a confined and mechanically ventilated enclosure fire. Fire Safety Journal. July 2017;91:291-302 EP002715. Hassi R'Mel; 2010 1997;33(2):144-156 2016 136 ## Edited by Fahmina Zafar and Anujit Ghosal To ensure a healthy lifestyle, fire safety and protocols are essential. The population boom, economic crunches, and excessive exploitation of nature have enhanced the possibilities of destruction due to an event of a fire. Computational simulations enacting case studies and incorporation of fire safety protocols in daily routines can help in avoiding such mishaps. Published in London, UK © 2020 IntechOpen © stockphoto / iStock Fire Safety and Management Awareness Fire Safety and Management Awareness Edited by Fahmina Zafar and Anujit Ghosal	doab	2025-04-07T03:56:59.092728	20-4-2021 18:24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "006df1b2-f518-4d6b-8d96-484202f13086", "url": "https://mts.intechopen.com/storage/books/10032/authors_book/authors_book.pdf", "author": "", "title": "Fire Safety and Management Awareness", "publisher": "IntechOpen", "isbn": "9781839624261", "section_idx": 9 }
0071a6c6-eff7-449d-a548-4af9bff79a91.0	Edited by Haiping Wang Cucumber is a well-known and popular vegetable because of its rich nutrient profile and versatile uses in culinary, therapeutic and cosmetic purposes. This book provides information on the plant's origins, biology, and breeding as well as research on its economic value, utilization, cultivation, and therapeutic benefits. ISBN 978-1-83968-023-6 Cucumber Economic Values and Its Cultivation and Breeding Published in London, UK © 2021 IntechOpen © midori4 / iStock	doab	2025-04-07T03:56:59.108892	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 0 }
0071a6c6-eff7-449d-a548-4af9bff79a91.1	Cucumber Economic Values and Its Cultivation and Breeding Edited by Haiping Wang	doab	2025-04-07T03:56:59.109012	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 1 }
0071a6c6-eff7-449d-a548-4af9bff79a91.2	Cucumber Economic Values and Its Cultivation and Breeding Edited by Haiping Wang Published in London, United Kingdom	doab	2025-04-07T03:56:59.109043	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 2 }
0071a6c6-eff7-449d-a548-4af9bff79a91.3	Supporting open minds since 2005 Cucumber Economic Values and Its Cultivation and Breeding http://dx.doi.org/10.5772/intechopen.87508 Edited by Haiping Wang #### Contributors Maadh Abdulwahab Alfahad, Gograj Singh Jat, Tusar Behera, Suman lata, Sachin Kumar, Olumide Samuel Daramola, Shampa Chakraborty, Sadhana Rayalu, Ravi Mohan Srivastava, Chidiebere Ugwu, Stephen Suru, Iyabo Bosede Adeoye, Sarmad Ghazi Al-Shawi, Sadiq Jaafir Aziz Alneamah, Oluwayemisi Ibitoye, Taofeek Ajiboye, Azeemat Titilola Abdulazeez, Rukayat Abiodun Oyegoke, Hamdalat Folake Muritala, Fatimah Aluko Abubakar, Bernard Ndubuisi Okafor, Japhet J. Yaduma, Olawale Arogundade, Titilayo Ajose, Joshua Matthew, Itunu Osijo, Michael Henry Henry Böhme, Haiping Wang, Huixia Jia, Sneha Joshih #### © The Editor(s) and the Author(s) 2021 The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be found at http://www.intechopen.com/copyright-policy.html. #### Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in London, United Kingdom, 2021 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, 5 Princes Gate Court, London, SW7 2QJ, United Kingdom Printed in Croatia British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Additional hard and PDF copies can be obtained from [email protected] Cucumber Economic Values and Its Cultivation and Breeding Edited by Haiping Wang p. cm. Print ISBN 978-1-83968-023-6 Online ISBN 978-1-83968-024-3 eBook (PDF) ISBN 978-1-83968-025-0	doab	2025-04-07T03:56:59.109069	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 3 }
0071a6c6-eff7-449d-a548-4af9bff79a91.4	We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists 5,500+ Open access books available 134,000+ International authors and editors 165M+ Downloads 156 Countries delivered to Our authors are among the Top 1% most cited scientists 12.2% Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)	doab	2025-04-07T03:56:59.109296	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 4 }
0071a6c6-eff7-449d-a548-4af9bff79a91.5	Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com	doab	2025-04-07T03:56:59.109361	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 5 }
0071a6c6-eff7-449d-a548-4af9bff79a91.6	Meet the editor Haiping Wang is a full-time research scientist and professor in the Department of Vegetables Germplasm, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVFCAAS). His research interests include vegetable genetic resources and preservation of the diversity of Midterm Gene-Bank of Vegetables Genetic Resources in China. Research on vegetable genetics is conducted to improve the crop for growers and consumers. His key areas of interest include garlic, ginger, radish, and cucumber genetics and the development of genomic tools. His outreach activities include interaction with the garlic and ginger production and with consumers. Dr. Wang is the author and co-author of seventy publications in scientific journals and thirteen book chapters in Chinese and English. He has reviewed numerous publications for more than ten international scientific journals. Contents Section 1 Section 2 Introductory Chapter: Studies on Cucumber Antidiabetic Principle in Cucumis sativus L. Cucumber Pickles and Fermentations by Chidiebere Ugwu and Stephen Suru (Cucumis sativus L.) by Iyabo Bosede Adeoye Section 3 Oyo State by Ibitoye Oluwayemisi Beatrice, Ajiboye Taofeek Olakunle, Abdulazeez Azeemat Titilola, Oyegoke Rukayat Abiodun, Muritala Hamdalat Folake and Abubakar Fatimah Aluko by Sarmad Ghazi Al-Shawi and Sadiq Jaafir Aziz Alneamah Cosmetic, Culinary and Therapeutic Uses of Cucumber Market Performance and Structure of Cucumber in Ibadan, by Huixia Jia and Haiping Wang Preface III Introduction 1 Chapter 1 3 Economic Values and Pharmacological Potentials 11 Chapter 2 13 Chapter 3 23 Chapter 4 39 Production and Market 49 Chapter 5 51	doab	2025-04-07T03:56:59.109394	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 6 }
0071a6c6-eff7-449d-a548-4af9bff79a91.7	Contents Preface Cucumber is an economically important fruit vegetable. With abundant water, nutrients and phytochemical composition, cucumber has versatile uses in culinary, therapeutic and cosmetic purposes. Cucumber has also multiple advantages such as being a diploid and having a small genome, short life cycle and self-compatible mating system, so it has been identified as a model plant for genetic studies. The scientific research of cucumber will be beneficial for its efficient protection and Cucumber was cultivated about 3000 years ago. It is indigenous to India and likely originated from the foothills of the Himalayas, and spread to Western Asia and Southern Europe. Cucumber was introduced respectively to North China through the Silk Route and to South China from Burma and the India–China border, and subsequently spread to East Asia. Nowadays, cucumber is widely cultivated in Over six sections, this book presents the latest findings and research on several aspects of cucumber biology, breeding, cultivation, marketing, and more. Section 1, "Introduction," provides an overview of cucumber. Section 2, "Economic Values and Pharmacological Potentials," helps readers to understand the nutrients, functional components, and extensive utilization of cucumber. Section 3, "Production and Market," explains the market structure and earning situation related to selling cucumber. Section 4 "Cultivation and Management" and Section 5 "Pest and Disease Prevention" discusses the cultivation and improvement of cucumber to obtain high yield. Section 6, "Breeding Progress," summarizes the classical genetics and traditional breeding of cucumber, as well as presents prospects for future I would like to extend thanks to all authors who contributed to this book. My sincere thanks go also Author Service Manager Ms. Jasna Bozic at IntechOpen who helped Haiping Wang Beijing, China Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, me through all the stages of preparing and publishing this book. temperate and tropical regions throughout the world. utilization. breeding.	doab	2025-04-07T03:56:59.109540	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 7 }
0071a6c6-eff7-449d-a548-4af9bff79a91.8	Preface Cucumber is an economically important fruit vegetable. With abundant water, nutrients and phytochemical composition, cucumber has versatile uses in culinary, therapeutic and cosmetic purposes. Cucumber has also multiple advantages such as being a diploid and having a small genome, short life cycle and self-compatible mating system, so it has been identified as a model plant for genetic studies. The scientific research of cucumber will be beneficial for its efficient protection and utilization. Cucumber was cultivated about 3000 years ago. It is indigenous to India and likely originated from the foothills of the Himalayas, and spread to Western Asia and Southern Europe. Cucumber was introduced respectively to North China through the Silk Route and to South China from Burma and the India–China border, and subsequently spread to East Asia. Nowadays, cucumber is widely cultivated in temperate and tropical regions throughout the world. Over six sections, this book presents the latest findings and research on several aspects of cucumber biology, breeding, cultivation, marketing, and more. Section 1, "Introduction," provides an overview of cucumber. Section 2, "Economic Values and Pharmacological Potentials," helps readers to understand the nutrients, functional components, and extensive utilization of cucumber. Section 3, "Production and Market," explains the market structure and earning situation related to selling cucumber. Section 4 "Cultivation and Management" and Section 5 "Pest and Disease Prevention" discusses the cultivation and improvement of cucumber to obtain high yield. Section 6, "Breeding Progress," summarizes the classical genetics and traditional breeding of cucumber, as well as presents prospects for future breeding. I would like to extend thanks to all authors who contributed to this book. My sincere thanks go also Author Service Manager Ms. Jasna Bozic at IntechOpen who helped me through all the stages of preparing and publishing this book. > Haiping Wang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China 1 Section 1 Introduction Section 1 Introduction 3 Chapter 1 1. Introduction progress of cucumber. 2. Biological characteristics seeds. The weight of 1000 seeds is about 20–40 g. on Cucumber Huixia Jia and Haiping Wang Introductory Chapter: Studies Cucumber (Cucumis sativus L.) belongs to Cucumis genus in Cucurbitaceae family and is an economically important fruit vegetable. There are three wild or semi-wild varieties of cucumber: C. sativus L. var. hardwickii, C. sativus L. var. sikkimensis, C. sativus L. var. xishuangbannanesis. Cucumber is indigenous to India and likely originated from the foothills of the Himalayan Mountain [1, 2]. Cucumber was cultivated ~3000 years ago in India, and it seems to spread rapidly to Western Asia, and then to Southern Europe [2]. Cucumber was introduced respectively to North China through the Silk Route and to South China from Burma and India-China border, and subsequently spread to East Asia [2]. Genome variation analysis showed cucumber core germplasms were divided into four geographic groups including India, Eurasia, East Asia, and Xishuangbanna [3]. Nowadays, cucumber is widely cultivated in temperate and tropical regions throughout the world [4]. The total production of cucumber was 87,805,086 tons worldwide, and Asia was the largest producer accounting for 84.9% of the world's total production in 2019 (www.fao. org/faostat/en/). With abundant water, nutrients and phytochemical composition, cucumber has versatile uses in culinary, therapeutic and cosmetic purposes [5, 6]. Cucumber has multiple advantages such as diploid, small genome, short life cycle and self-compatible mating system, so it is suitable for genetic studies. Moreover, cucumber has been identified as a model plant for studying sex determination and plant vascular biology [7]. Consequently, numerous studies have been conducted to discover the miracle of cucumber. The book will cover the extensive benefits, production and market, cultivation and management, pests and diseases, breeding Cucumber is an annual climbing herbaceous plant. The root system is shallow and mainly distributes in the cultivated land layer of 30 cm. The stem is vine with different degree of apical dominance. The cross section of the stem is rhombus, and the epidermis of the stem has burrs. The axillae on the stem have the ability of branching, and the number of branching varies greatly among varieties. The cotyledons of cucumber are opposite and long elliptic; euphylla are alternate, simple, pentagonal palmate or cordate in outline, and the blades are 3–7 lobed. The flower is axillary, unisexual and occasionally hermaphrodite. The calyx is green with bristles, and the corolla is yellow. The colour of young fruit changes from white to pale green, while mature fruit is yellow or brown when ripened. The shape of the fruit is diverse, such as clublike, cylindrical and spherical. Each fruit has 100–400	doab	2025-04-07T03:56:59.109712	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 8 }
0071a6c6-eff7-449d-a548-4af9bff79a91.10	Introductory Chapter: Studies on Cucumber Huixia Jia and Haiping Wang	doab	2025-04-07T03:56:59.109916	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 10 }
0071a6c6-eff7-449d-a548-4af9bff79a91.11	1. Introduction Cucumber (Cucumis sativus L.) belongs to Cucumis genus in Cucurbitaceae family and is an economically important fruit vegetable. There are three wild or semi-wild varieties of cucumber: C. sativus L. var. hardwickii, C. sativus L. var. sikkimensis, C. sativus L. var. xishuangbannanesis. Cucumber is indigenous to India and likely originated from the foothills of the Himalayan Mountain [1, 2]. Cucumber was cultivated ~3000 years ago in India, and it seems to spread rapidly to Western Asia, and then to Southern Europe [2]. Cucumber was introduced respectively to North China through the Silk Route and to South China from Burma and India-China border, and subsequently spread to East Asia [2]. Genome variation analysis showed cucumber core germplasms were divided into four geographic groups including India, Eurasia, East Asia, and Xishuangbanna [3]. Nowadays, cucumber is widely cultivated in temperate and tropical regions throughout the world [4]. The total production of cucumber was 87,805,086 tons worldwide, and Asia was the largest producer accounting for 84.9% of the world's total production in 2019 (www.fao. org/faostat/en/). With abundant water, nutrients and phytochemical composition, cucumber has versatile uses in culinary, therapeutic and cosmetic purposes [5, 6]. Cucumber has multiple advantages such as diploid, small genome, short life cycle and self-compatible mating system, so it is suitable for genetic studies. Moreover, cucumber has been identified as a model plant for studying sex determination and plant vascular biology [7]. Consequently, numerous studies have been conducted to discover the miracle of cucumber. The book will cover the extensive benefits, production and market, cultivation and management, pests and diseases, breeding progress of cucumber.	doab	2025-04-07T03:56:59.110030	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 11 }
0071a6c6-eff7-449d-a548-4af9bff79a91.12	2. Biological characteristics Cucumber is an annual climbing herbaceous plant. The root system is shallow and mainly distributes in the cultivated land layer of 30 cm. The stem is vine with different degree of apical dominance. The cross section of the stem is rhombus, and the epidermis of the stem has burrs. The axillae on the stem have the ability of branching, and the number of branching varies greatly among varieties. The cotyledons of cucumber are opposite and long elliptic; euphylla are alternate, simple, pentagonal palmate or cordate in outline, and the blades are 3–7 lobed. The flower is axillary, unisexual and occasionally hermaphrodite. The calyx is green with bristles, and the corolla is yellow. The colour of young fruit changes from white to pale green, while mature fruit is yellow or brown when ripened. The shape of the fruit is diverse, such as clublike, cylindrical and spherical. Each fruit has 100–400 seeds. The weight of 1000 seeds is about 20–40 g.	doab	2025-04-07T03:56:59.110077	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 12 }
0071a6c6-eff7-449d-a548-4af9bff79a91.13	3. Culinary, therapeutic and cosmetic uses of cucumber At present, cucumber is the fourth most widely cultivated vegetable after tomato, cabbage and onion [8]. Cucumber has versatile uses in culinary, therapeutic and cosmetic purposes [5, 6]. Nutritional and epidemiological researches have shown various benefits of cucumber. For example, cucumber contains abundant nutrients and has crunchy texture and unique flavor, so it is a quintessential vegetable used for a variety of dishes, and it is also indispensable for salad, soup and smoothie. Cucumber is rich in superior hydration and phytochemicals, which have diverse health benefits including weight loss, anti-inflammation, remedy for multiple diseases of eczema, constipation, hypertension, atherosclerosis, cancer, etc. [9]. Recent studies found that the presence of kaempferol in cucumber is an important antidiabetic agent [10]. Furthermore, cucumber is popularly used for natural beautification and for skin treatments [11].	doab	2025-04-07T03:56:59.110113	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 13 }
0071a6c6-eff7-449d-a548-4af9bff79a91.14	4. Influence factors and solutions of cucumber fermentation Cucumber pickles are most commonly fermented vegetable and widely consumed throughout the world [12]. Good fermentation depends on the proper combinations and interactions of multiple physical, chemical and microbiological factors [13]. Brine storage and process operations are susceptible to oxidation reactions during the fermentation process, and this has adverse influence on the quality property of cucumber pickles. To control the influence factors of cucumber fermentation, researchers have done many efforts on modern and advanced technologies, such as reducing the concentration of brining sodium chloride, developing the brining properties using lactic acid bacteria cultures, developing an anaerobic tank system, preventing cucumber gaseous deterioration by pouring of CO2 from fermentation brines [13]. After storing the brine, excess salt and organic wastes need to be leached to complete the product processing, and these wastes are sources of serious environmental concern. Thus, the waste disposal needs to be solved in the cucumber pickle industry. #### 5. Performance, structure and constraints of cucumber market Marketing is vital for linking production and consumption and facilitating agricultural productivity and employment [14]. Market performance is the ultimate result of various market activities, and market structure is the organization characteristics of the market that influence the nature of competition and pricing [15]. Both male and female participate in cucumber marketing, and the male–female rate has great differences in different regions. The wholesalers are older than the retailers. In Ibadan, most of the retailers were within 31–40 years age, whereas most of the wholesalers were within 41–50 years age. It's gratifying that cucumber marketing is usually profitable for the retailers and wholesalers at both peak and lean seasons of cucumber production. However, the cucumber market is competitive, and inequality exists in the market. Commodity perishability is an important constraint in cucumber market. Thus, it is indispensable to reduce perishable degree and prolong storage time after post-harvest. 5 Introductory Chapter: Studies on Cucumber DOI: http://dx.doi.org/10.5772/intechopen.97360 cultivated condition biostimulator is applied. 6. Soil moisture and fertilizer management of cucumber Inappropriate farming systems and poor agronomic management are 7. Biostimulators promote growth of cucumber under soilless 8. Pests and diseases during cucumber cultivation and production During growth process, cucumber might be affected by multiple insect pests and diseases, resulting in decrease of yield and quality. The major insect pests in cucumber including Diabrotica undecimpunctata, Acalymma vitatum, Bactrocera cucurbitae, Raphidopalpa foveicollis, Epilachna implicate, Myzus persicae, Aphis gossypii, Anasa tristis, Trialeurodes vaparariorus, Bemisia tabaci and B. argentifolii [22, 23]. Currently, the pest management mainly relies on chemical pesticides Soilless cultivation in substrate culture is an important cultivation pattern for cucumber in greenhouses. The substrates should have specific physical properties including pore volume, air and water capacity, and density of substrates. Studies indicate that biostimulators can stabilize the production process to enhance plant growth under stress conditions. For instance, humate can increase vitality and growth of plants, improve seed germination, promote nutrient uptake, enhance transport and availability of micronutrients, and increase ion-exchange capacity. Lactates can produce bioregulatory effects to improve nutrient balance and plant vitality [21]. Bacillus subtilis, as a microorganism from the rhizosphere, can accelerate plant growth, stimulate the process of formation of plant organs, and enhance the resistance of biotic and abiotic stresses [21]. Application of biostimulators mixture (humate, lactate, and Bacillus subtilis) prevent growth reduction of cucumber under pH and temperature stresses through enhancing the root growth, whereas the growth is markedly reduced under stresses if no responsible for low yield of cucumber. The quality/fertility status of soils is essential for growth and development for cucumber [16]. With good moisture and fertilizer management, optimum yield of cucumber might be attained. The conventional irrigation methods including flooding irrigation, furrow irrigation and drip irrigation have been widely applied for a long time in cucumber cultivation because of their low cost or simple operation [17, 18]. However, these irrigation methods are surface irrigation and are driven by positive pressure, which may cause low water use efficiency, water wastage and nutrient loss [16, 19]. To solve these problems, new irrigation technique such as negative pressure irrigation that controls automatically water release based on the soil water potential difference should be encouraged [16]. Inadequate fertilizer use causes low soil fertility that cannot provide sufficient nutrients for the normal growth of cucumber. The integration application of inorganic and organic fertilizer is more beneficial than the sole use of inorganic fertilizer or organic manures in cucumber production [20]. Moreover, fertilizer sources need to dissolve or decompose to make nutrients available for cucumber plants, so soil fertility also depends on soil water, temperature and density. Consequently, the soil management strategies such as negative pressure irrigation, seasonable fertilization, application of organic mulches and conservation tillage should be appropriately applied for sustainable production of cucumber. Cucumber Economic Values and Its Cultivation and Breeding treatments [11]. industry. 3. Culinary, therapeutic and cosmetic uses of cucumber At present, cucumber is the fourth most widely cultivated vegetable after tomato, cabbage and onion [8]. Cucumber has versatile uses in culinary, therapeutic and cosmetic purposes [5, 6]. Nutritional and epidemiological researches have shown various benefits of cucumber. For example, cucumber contains abundant nutrients and has crunchy texture and unique flavor, so it is a quintessential vegetable used for a variety of dishes, and it is also indispensable for salad, soup and smoothie. Cucumber is rich in superior hydration and phytochemicals, which have diverse health benefits including weight loss, anti-inflammation, remedy for multiple diseases of eczema, constipation, hypertension, atherosclerosis, cancer, etc. [9]. Recent studies found that the presence of kaempferol in cucumber is an important antidiabetic agent [10]. Furthermore, cucumber is popularly used for natural beautification and for skin 4. Influence factors and solutions of cucumber fermentation 5. Performance, structure and constraints of cucumber market agricultural productivity and employment [14]. Market performance is the ultimate result of various market activities, and market structure is the are older than the retailers. In Ibadan, most of the retailers were within 31–40 years age, whereas most of the wholesalers were within 41–50 years age. It's gratifying that cucumber marketing is usually profitable for the retailers and wholesalers at both peak and lean seasons of cucumber production. However, the cucumber market is competitive, and inequality exists in the market. Commodity perishability is an important constraint in cucumber market. Thus, it is indispensable to reduce perishable degree and prolong storage time after Marketing is vital for linking production and consumption and facilitating organization characteristics of the market that influence the nature of competition and pricing [15]. Both male and female participate in cucumber marketing, and the male–female rate has great differences in different regions. The wholesalers Cucumber pickles are most commonly fermented vegetable and widely consumed throughout the world [12]. Good fermentation depends on the proper combinations and interactions of multiple physical, chemical and microbiological factors [13]. Brine storage and process operations are susceptible to oxidation reactions during the fermentation process, and this has adverse influence on the quality property of cucumber pickles. To control the influence factors of cucumber fermentation, researchers have done many efforts on modern and advanced technologies, such as reducing the concentration of brining sodium chloride, developing the brining properties using lactic acid bacteria cultures, developing an anaerobic tank system, preventing cucumber gaseous deterioration by pouring of CO2 from fermentation brines [13]. After storing the brine, excess salt and organic wastes need to be leached to complete the product processing, and these wastes are sources of serious environmental concern. Thus, the waste disposal needs to be solved in the cucumber pickle 4 post-harvest.	doab	2025-04-07T03:56:59.110198	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 14 }
0071a6c6-eff7-449d-a548-4af9bff79a91.15	6. Soil moisture and fertilizer management of cucumber Inappropriate farming systems and poor agronomic management are responsible for low yield of cucumber. The quality/fertility status of soils is essential for growth and development for cucumber [16]. With good moisture and fertilizer management, optimum yield of cucumber might be attained. The conventional irrigation methods including flooding irrigation, furrow irrigation and drip irrigation have been widely applied for a long time in cucumber cultivation because of their low cost or simple operation [17, 18]. However, these irrigation methods are surface irrigation and are driven by positive pressure, which may cause low water use efficiency, water wastage and nutrient loss [16, 19]. To solve these problems, new irrigation technique such as negative pressure irrigation that controls automatically water release based on the soil water potential difference should be encouraged [16]. Inadequate fertilizer use causes low soil fertility that cannot provide sufficient nutrients for the normal growth of cucumber. The integration application of inorganic and organic fertilizer is more beneficial than the sole use of inorganic fertilizer or organic manures in cucumber production [20]. Moreover, fertilizer sources need to dissolve or decompose to make nutrients available for cucumber plants, so soil fertility also depends on soil water, temperature and density. Consequently, the soil management strategies such as negative pressure irrigation, seasonable fertilization, application of organic mulches and conservation tillage should be appropriately applied for sustainable production of cucumber.	doab	2025-04-07T03:56:59.110735	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 15 }
0071a6c6-eff7-449d-a548-4af9bff79a91.16	7. Biostimulators promote growth of cucumber under soilless cultivated condition Soilless cultivation in substrate culture is an important cultivation pattern for cucumber in greenhouses. The substrates should have specific physical properties including pore volume, air and water capacity, and density of substrates. Studies indicate that biostimulators can stabilize the production process to enhance plant growth under stress conditions. For instance, humate can increase vitality and growth of plants, improve seed germination, promote nutrient uptake, enhance transport and availability of micronutrients, and increase ion-exchange capacity. Lactates can produce bioregulatory effects to improve nutrient balance and plant vitality [21]. Bacillus subtilis, as a microorganism from the rhizosphere, can accelerate plant growth, stimulate the process of formation of plant organs, and enhance the resistance of biotic and abiotic stresses [21]. Application of biostimulators mixture (humate, lactate, and Bacillus subtilis) prevent growth reduction of cucumber under pH and temperature stresses through enhancing the root growth, whereas the growth is markedly reduced under stresses if no biostimulator is applied.	doab	2025-04-07T03:56:59.110780	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 16 }
0071a6c6-eff7-449d-a548-4af9bff79a91.17	8. Pests and diseases during cucumber cultivation and production During growth process, cucumber might be affected by multiple insect pests and diseases, resulting in decrease of yield and quality. The major insect pests in cucumber including Diabrotica undecimpunctata, Acalymma vitatum, Bactrocera cucurbitae, Raphidopalpa foveicollis, Epilachna implicate, Myzus persicae, Aphis gossypii, Anasa tristis, Trialeurodes vaparariorus, Bemisia tabaci and B. argentifolii [22, 23]. Currently, the pest management mainly relies on chemical pesticides that cause environmental pollution, pest resistance, and disturbance of balance between the pests and natural enemies. Moreover, this control strategy is harmful to human health. Therefore, an integrated pest management including pest monitoring, cultural method, host resistance, botanicals, biological control, and judicious use of chemicals is recommended for controlling these pests [24, 25] Many diseases caused by viral, bacterial, fungal and nematode pathogens severely affect the cultivation and production of cucumber. Viruses infecting cucumber belong to three genera: Potyvirus, Cucumovirus and Crinivirus [26]. Especially, the CMV, ZYMV, WMV, MWMV, PRSV and BPYV are major viruses that cause severe symptoms to cucumber. Downy mildew, powdery mildew and anthracnose also cause substantial losses of cucumber production [27]. Some pathogenic fungi including Alternaria tenuis, Fusarium equisett, Phytophthora capsici, Botrytis cinerea and Cladosporium tenuissimum cause rotting and high post-harvest losses of cucumber [28]. Furthermore, root-knot nematodes are prevalent destructive pathogens of cucumber [29]. Though a series of chemicals have been evaluated and screened to control these diseases, the biological control strategy and high-resistant varieties of cucumber need be developed and created to resist diseases in efficient and environmental ways. ## 9. Polyphenols act as antioxidants in cucumber to defense stresses Plant secondary metabolites play important roles in adapting to various environments and defensing against biotic and abiotic stresses. Cucumber is a rich source of phenolic compounds that are important secondary metabolites [30, 31]. The antioxidant capacity of cucumber seems to be attributing to polyphenols that scavenge singlet oxygen, hydroxyl and lipid peroxyl radicals to prevent lipid oxidation. Better understanding of the molecular regulation of polyphenols biosynthesis is crucial to increase the production of polyphenols. Polyphenols are derivatives of phenylpropanoid pathway which involves an array of enzymes. Among these, phenylalanine ammonia lyase, chalcone synthase, cinnamate 4-hydroxylase and dihydroflavonol reductase play important roles [32]. In-depth study of these key enzymes in cucumber will facilitate to reveal the molecular mechanism of polyphenol synthesis, which is helpful for advancement in biotechnological and industrial applications. #### 10. Progress of traditional breeding and molecular breeding in cucumber In the past decades, traditional breeding has played essential roles in cultivar innovation of cucumber. Some superior varieties with early maturity, high yield and high resistance have been developed through hybridization and mutagenesis [33]. However, this progress is slow because of the long cycle and difficulty in selection of stable genetic characters or genotypes. To overcome the obstacle of traditional breeding, molecular breeding technologies including molecular marker assisted breeding, genome-wide design breeding and genetic engineering have been applied in cucumber to accelerate the breeding cycle and select desirable traits. Molecular breeding of cucumber has made some progress and achievements on completion of genomics, genetic architecture and molecular mechanism underlying important traits, and creation of high quality and multi-resistant varieties [7, 34–36]. With increasing consumption demand of cucumber, more new varieties with excellent comprehensive properties are in need, and we might make some efforts from the following aspects: (i) expanding collection and utilization of cucumber germplasm 7 Author details Beijing, China Huixia Jia\* and Haiping Wang\* provided the original work is properly cited. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, \Address all correspondence to: [email protected] and [email protected] © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, Introductory Chapter: Studies on Cucumber DOI: http://dx.doi.org/10.5772/intechopen.97360* and phenomics [37]. resources; (ii) establishing highly efficient gene editing and genetic transformation technologies in cucumber; (iii) identifying new loci or genes associated with key agronomic traits of cucumber and combining multiple molecular markers of excellent traits into one variety; (iv) realizing rapid accumulation of omics genotypes #### Introductory Chapter: Studies on Cucumber DOI: http://dx.doi.org/10.5772/intechopen.97360 Cucumber Economic Values and Its Cultivation and Breeding and environmental ways. biotechnological and industrial applications. that cause environmental pollution, pest resistance, and disturbance of balance between the pests and natural enemies. Moreover, this control strategy is harmful to human health. Therefore, an integrated pest management including pest monitoring, cultural method, host resistance, botanicals, biological control, and judicious use of chemicals is recommended for controlling these pests [24, 25] Many diseases caused by viral, bacterial, fungal and nematode pathogens severely affect the cultivation and production of cucumber. Viruses infecting cucumber belong to three genera: Potyvirus, Cucumovirus and Crinivirus [26]. Especially, the CMV, ZYMV, WMV, MWMV, PRSV and BPYV are major viruses that cause severe symptoms to cucumber. Downy mildew, powdery mildew and anthracnose also cause substantial losses of cucumber production [27]. Some pathogenic fungi including Alternaria tenuis, Fusarium equisett, Phytophthora capsici, Botrytis cinerea and Cladosporium tenuissimum cause rotting and high post-harvest losses of cucumber [28]. Furthermore, root-knot nematodes are prevalent destructive pathogens of cucumber [29]. Though a series of chemicals have been evaluated and screened to control these diseases, the biological control strategy and high-resistant varieties of cucumber need be developed and created to resist diseases in efficient 9. Polyphenols act as antioxidants in cucumber to defense stresses Plant secondary metabolites play important roles in adapting to various environments and defensing against biotic and abiotic stresses. Cucumber is a rich source of phenolic compounds that are important secondary metabolites [30, 31]. The antioxidant capacity of cucumber seems to be attributing to polyphenols that scavenge singlet oxygen, hydroxyl and lipid peroxyl radicals to prevent lipid oxidation. Better understanding of the molecular regulation of polyphenols biosynthesis is crucial to increase the production of polyphenols. Polyphenols are derivatives of phenylpropanoid pathway which involves an array of enzymes. Among these, phenylalanine ammonia lyase, chalcone synthase, cinnamate 4-hydroxylase and dihydroflavonol reductase play important roles [32]. In-depth study of these key enzymes in cucumber will facilitate to reveal the molecular mechanism of polyphenol synthesis, which is helpful for advancement in 10. Progress of traditional breeding and molecular breeding in cucumber In the past decades, traditional breeding has played essential roles in cultivar innovation of cucumber. Some superior varieties with early maturity, high yield and high resistance have been developed through hybridization and mutagenesis [33]. However, this progress is slow because of the long cycle and difficulty in selection of stable genetic characters or genotypes. To overcome the obstacle of traditional breeding, molecular breeding technologies including molecular marker assisted breeding, genome-wide design breeding and genetic engineering have been applied in cucumber to accelerate the breeding cycle and select desirable traits. Molecular breeding of cucumber has made some progress and achievements on completion of genomics, genetic architecture and molecular mechanism underlying important traits, and creation of high quality and multi-resistant varieties [7, 34–36]. With increasing consumption demand of cucumber, more new varieties with excellent comprehensive properties are in need, and we might make some efforts from the following aspects: (i) expanding collection and utilization of cucumber germplasm 6 resources; (ii) establishing highly efficient gene editing and genetic transformation technologies in cucumber; (iii) identifying new loci or genes associated with key agronomic traits of cucumber and combining multiple molecular markers of excellent traits into one variety; (iv) realizing rapid accumulation of omics genotypes and phenomics [37].	doab	2025-04-07T03:56:59.110818	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 17 }
0071a6c6-eff7-449d-a548-4af9bff79a91.18	Author details Huixia Jia\* and Haiping Wang\* Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China \*Address all correspondence to: [email protected] and [email protected] © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.	doab	2025-04-07T03:56:59.111233	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 18 }
0071a6c6-eff7-449d-a548-4af9bff79a91.20	Economic Values and Pharmacological Potentials Cucumber Economic Values and Its Cultivation and Breeding HPLC–ESI-Q-TOF-MS for a comprehensive characterization of bioactive phenolic compounds in cucumber whole fruit extract. Food Research International. 2012;46(1):108-117. [31] Murcia MA, Jiménez AM, Martínez-Tomé M. Vegetables antioxidant losses during industrial processing and refrigerated storage. Food Research International. 2009;42(8):1046-1052. [32] Peukert M, Weise S, Röder MS, Matthies IE. Development of SNP markers for genes of the phenyl propanoid pathway and their association to kernel and malting traits in barley. BMC Genetics. 2013;14:97. [33] Feng SJ, Zhang JP, Mu ZH, Wang YJ, Wen CL, Wu T, et al. Recent progress on the molecular breeding of Cucumis sativus L. in China. Theoretical and Applied Genetics. 2020;133:1777-1790. [34] Wang YH, Bo KL, Gu XF, Pan JS, Li YH, Chen JF, et al. Molecularly tagged genes and quantitative trait loci in cucumber with recommendations for QTL nomenclature. Horticulture [35] Wang YH, Jiang B, Dymerski R, Xu XW, Weng YQ. Quantitative trait loci for horticulturally important traits defining the Sikkim cucumber, Cucumis sativus var. sikkimensis. Theoretical and Applied Genetics. 2020;134(1):1-19. [36] Zhang ZH, Map LY, Chen HM, Bu FJ, Li GC, Sun JJ, et al. Genome-wide mapping of structural variations reveals a copy number variant that determines reproductive morphology in cucumber. Plant Cell. 2015;27(6):1595-1604. [37] Feng SJ, Zhang JP, Mu ZH, Wang YJ, Wen CL, Wu T, et al. Recent progress on the molecular breeding of Cucumis sativus L. in China. Theoretical and Applied Genetics. 2020;133(7): Research. 2020;7:3. 10 1777-1790. 13 Chapter 2 Abstract activity of C. sativus. 1. Introduction activity [11]. Antidiabetic Principle in Keywords: diabetes, Cucumis sativus, flavonoids, kaempferol Diabetes caused 4.2 million deaths in 2019 [2]. cardiovascular complications [9], and ulcerations [10]. Ibitoye Oluwayemisi Beatrice, Ajiboye Taofeek Olakunle, Abdulazeez Azeemat Titilola, Oyegoke Rukayat Abiodun, Muritala Hamdalat Folake and Abubakar Fatimah Aluko Diabetes is one of the leading cause of death globally. One of the strategies towards managing diabetes is the antidiabetic drugs which has recorded a huge success but accompanied with different degrees of side effect, hence, the use of natural plants products is encouraged. Several reports of antidiabetic medicinal plants have flooded literature but few has led to identification of active ingredient in such. Cucumis sativus is one of such plants reported to have antidiabetic property but there is little or no data on the active agent. This chapter therefore provides report on the active principle and mechanism of action underlying the antidiabetic Diabetes is a disorder where the body cells cannot use glucose effectively due to low insulin (Type 1 diabetes) or insulin insensitivity (Type 2 diabetes), therefore the blood glucose level increases [1]. It is characterized by a fasting blood glucose level higher than 126 mg/dL. It is one of the top 10 causes of death globally. About 463 million adults are living with diabetes; by 2045 this will rise to 700 million and In 2017, total estimated cost of diagnosed diabetes in the U.S. was \$327 billion [3]. Some complications of diabetes are oxidative stress, dyslipidaemia, endoplasmic The management of diabetes has involved many approaches in order to enhance the availability of insulin, boost insulin sensitivity and reduce alpha glucosidase From research, people with excess weight can greatly manage diabetes by engag- reticulum (ER) stress [4, 5], retinopathy [6], neuropathy [7], nephropathy [8], ing in moderate and considerate weight loss plan, also exercise can help control blood sugar levels, reduce glycated hemoglobin and reduce insulin resistance. Antidiabetic drugs are pharmacological substances that are employed in treating hyperglycemia when life style modifications do not bring desired effects [12]. They are categorized into different classes and they work either to enhance synthesis of insulin or reduce blood glucose level using different strategies. Cucumis sativus L.	doab	2025-04-07T03:56:59.111281	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 20 }
0071a6c6-eff7-449d-a548-4af9bff79a91.22	Antidiabetic Principle in Cucumis sativus L. Ibitoye Oluwayemisi Beatrice, Ajiboye Taofeek Olakunle, Abdulazeez Azeemat Titilola, Oyegoke Rukayat Abiodun, Muritala Hamdalat Folake and Abubakar Fatimah Aluko	doab	2025-04-07T03:56:59.111572	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 22 }
0071a6c6-eff7-449d-a548-4af9bff79a91.23	Abstract Diabetes is one of the leading cause of death globally. One of the strategies towards managing diabetes is the antidiabetic drugs which has recorded a huge success but accompanied with different degrees of side effect, hence, the use of natural plants products is encouraged. Several reports of antidiabetic medicinal plants have flooded literature but few has led to identification of active ingredient in such. Cucumis sativus is one of such plants reported to have antidiabetic property but there is little or no data on the active agent. This chapter therefore provides report on the active principle and mechanism of action underlying the antidiabetic activity of C. sativus. Keywords: diabetes, Cucumis sativus, flavonoids, kaempferol #### 1. Introduction Diabetes is a disorder where the body cells cannot use glucose effectively due to low insulin (Type 1 diabetes) or insulin insensitivity (Type 2 diabetes), therefore the blood glucose level increases [1]. It is characterized by a fasting blood glucose level higher than 126 mg/dL. It is one of the top 10 causes of death globally. About 463 million adults are living with diabetes; by 2045 this will rise to 700 million and Diabetes caused 4.2 million deaths in 2019 [2]. In 2017, total estimated cost of diagnosed diabetes in the U.S. was \$327 billion [3]. Some complications of diabetes are oxidative stress, dyslipidaemia, endoplasmic reticulum (ER) stress [4, 5], retinopathy [6], neuropathy [7], nephropathy [8], cardiovascular complications [9], and ulcerations [10]. The management of diabetes has involved many approaches in order to enhance the availability of insulin, boost insulin sensitivity and reduce alpha glucosidase activity [11]. From research, people with excess weight can greatly manage diabetes by engaging in moderate and considerate weight loss plan, also exercise can help control blood sugar levels, reduce glycated hemoglobin and reduce insulin resistance. Antidiabetic drugs are pharmacological substances that are employed in treating hyperglycemia when life style modifications do not bring desired effects [12]. They are categorized into different classes and they work either to enhance synthesis of insulin or reduce blood glucose level using different strategies. These antidiabetic drugs are very effective in treating hyperglycemia, but despite this success, there has been increased side effects accompanying their use. Therefore, there is increased search for antidiabetic agents from medicinal plants with little or no adverse effects. Experimental reports have validated the presence of antidiabetic substances in medicinal plants [13–15]. One of such plants reported to have antidiabetic property is Cucumis sativus. Saidu et al.'s study reported the hypoglycemic property of methanolic fruit pulp extract of Cucumis sativus [16]. This chapter focuses on the antidiabetic principle identified in Cucumis sativus L. ### 2. Flavonoids Flavonoids are a class of plants secondary metabolites made up of polyphenolic structures that contribute to the color and fragrance of fruits and flowers, therefore, they constitute a significant part of the human diet [17]. As a large class, flavonoids are subdivided into groups based on the structure of their carbon rings and these include flavanols, flavones, chalcones, flavonones, flavanonols and isoflavones. They are abundantly distributed in vegetables, fruits and some beverages. Flavonoids possess a wide range of health-promoting properties like the antioxidant effect, anti-carcinogenic, anti-inflammatory and antidiabetic capabilities. They display these properties by modulating the functions of some cellular enzymes as well as inhibition of different enzymes like lipo-oxygenase, cyclo-oxygenase, phosphoinositide 3-kinase and xanthine oxidase [18, 19]. Therefore, they are indispensable components in various pharmaceutical, cosmetics and medicinal applications. One of the flavonoids that possesses antidiabetic property is kaempferol. #### 3. Cucumis sativus L. Cucumis sativus L. also known as Cucumber is a creeping plant in the family Cucurbitaceae. It is a fruit native to India and widely cultivated around the world. It is consumed fresh in salads, fermented (pickles) and as cooked vegetable [20]. Figure 1. High pressure liquid chromatographic profiling of the antidiabetic principle derived from Cucumis sativus fruit juice. 15 Figure 2. Structure of Kaempferol. Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393 ethanol and slightly soluble in water. kaempferol from Cucumis sativus L. [26]. 5. Biosynthesis of kaempferol flavonoid called kaempferol using HPLC (Figure 1). 3-O-glucoside [28]. 4. Kaempferol Cucumis sativus L. as a fruit, in addition to its nutritional value, has been reported to have some biological activities as anti-aging [21], antioxidant [22], and antidiabetic [23]. These properties have been linked to the presence of some phytochemical substances detected in Cucumis sativus L. like cucurbitacins [24], ascorbic acid [25], cucumerin, apigenin [26], lutein [27], quercetin 3-O-glucoside and kaempferol Recent studies validated the presence of antidiabetic agents in Cucumis sativus L. Ibitoye et al [29] identified the antidiabetic agent in Cucumis sativus L. as a Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one also known as kaempferol-3, Figure 2) is a yellow crystalline flavonoid having a molecular weight of 286.23 with a melting point of 276–278 °C. It is soluble in hot It has been isolated from different parts of different plants. Yang et al. separated kaempferol and its derivatives from the methanolic crude extract of Neocheiropteris palmatopedata by repeated column chromatography, using a Sephadex LH-20 column [30]. Orhan et al. reported the bioactivity-guided fractionation of Calluna vulgaris and isolated kaempferol galactoside using successive column chromatography techniques [31]. Ibitoye et al. also reported the bioactivity guided isolation of Kaempferol and its derivatives are synthesized in plants by different types of enzymes. Kaempferol is synthesized by condensation of 4-coumaroyl-CoA with tripropionyl-CoA to produce naringenin chalcone, this reaction is catalyzed by chalcone synthase [32]. Naringenin chalcone is then converted into a flavanone called naringenin, which is thereafter hydroxylated by flavanone 3-dioxygenase to produce dihydrokaempferol [33]. Finally, the introduction of a double bond at the There is no much data on the pharmacokinetics of Kaempferol, however, flavonoids are extensively metabolized by the colonic microflora [34, 35]. C2-C3 position of dihydrokaempferol produces kaempferol. Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393 Cucumis sativus L. as a fruit, in addition to its nutritional value, has been reported to have some biological activities as anti-aging [21], antioxidant [22], and antidiabetic [23]. These properties have been linked to the presence of some phytochemical substances detected in Cucumis sativus L. like cucurbitacins [24], ascorbic acid [25], cucumerin, apigenin [26], lutein [27], quercetin 3-O-glucoside and kaempferol 3-O-glucoside [28]. Recent studies validated the presence of antidiabetic agents in Cucumis sativus L. Ibitoye et al [29] identified the antidiabetic agent in Cucumis sativus L. as a flavonoid called kaempferol using HPLC (Figure 1).	doab	2025-04-07T03:56:59.111601	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 23 }
0071a6c6-eff7-449d-a548-4af9bff79a91.24	4. Kaempferol Cucumber Economic Values and Its Cultivation and Breeding 2. Flavonoids 3. Cucumis sativus L. These antidiabetic drugs are very effective in treating hyperglycemia, but despite this success, there has been increased side effects accompanying their use. Therefore, there is increased search for antidiabetic agents from medicinal plants with little or no adverse effects. Experimental reports have validated the presence of antidiabetic substances in medicinal plants [13–15]. One of such plants reported to have antidiabetic property is Cucumis sativus. Saidu et al.'s study reported the hypoglycemic property of methanolic fruit pulp extract of Cucumis sativus [16]. This chapter focuses on the antidiabetic principle identified in Cucumis sativus L. Flavonoids are a class of plants secondary metabolites made up of polyphenolic structures that contribute to the color and fragrance of fruits and flowers, therefore, they constitute a significant part of the human diet [17]. As a large class, flavonoids are subdivided into groups based on the structure of their carbon rings and these include flavanols, flavones, chalcones, flavonones, flavanonols and isoflavones. They are abundantly distributed in vegetables, fruits and some beverages. Flavonoids possess a wide range of health-promoting properties like the antioxidant effect, anti-carcinogenic, anti-inflammatory and antidiabetic capabilities. They display these properties by modulating the functions of some cellular enzymes as well as inhibition of different enzymes like lipo-oxygenase, cyclo-oxygenase, phosphoinositide 3-kinase and xanthine oxidase [18, 19]. Therefore, they are indispensable components in various pharmaceutical, cosmetics and medicinal applications. One of the flavonoids that possesses antidiabetic property is kaempferol. Cucumis sativus L. also known as Cucumber is a creeping plant in the family Cucurbitaceae. It is a fruit native to India and widely cultivated around the world. It is consumed fresh in salads, fermented (pickles) and as cooked vegetable [20]. High pressure liquid chromatographic profiling of the antidiabetic principle derived from Cucumis sativus 14 Figure 1. fruit juice. Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one also known as kaempferol-3, Figure 2) is a yellow crystalline flavonoid having a molecular weight of 286.23 with a melting point of 276–278 °C. It is soluble in hot ethanol and slightly soluble in water. It has been isolated from different parts of different plants. Yang et al. separated kaempferol and its derivatives from the methanolic crude extract of Neocheiropteris palmatopedata by repeated column chromatography, using a Sephadex LH-20 column [30]. Orhan et al. reported the bioactivity-guided fractionation of Calluna vulgaris and isolated kaempferol galactoside using successive column chromatography techniques [31]. Ibitoye et al. also reported the bioactivity guided isolation of kaempferol from Cucumis sativus L. [26]. Figure 2. Structure of Kaempferol. #### 5. Biosynthesis of kaempferol Kaempferol and its derivatives are synthesized in plants by different types of enzymes. Kaempferol is synthesized by condensation of 4-coumaroyl-CoA with tripropionyl-CoA to produce naringenin chalcone, this reaction is catalyzed by chalcone synthase [32]. Naringenin chalcone is then converted into a flavanone called naringenin, which is thereafter hydroxylated by flavanone 3-dioxygenase to produce dihydrokaempferol [33]. Finally, the introduction of a double bond at the C2-C3 position of dihydrokaempferol produces kaempferol. There is no much data on the pharmacokinetics of Kaempferol, however, flavonoids are extensively metabolized by the colonic microflora [34, 35]. #### Figure 3. Biological roles of Kaempferol. Intestinal permeability study of kaempferol shows it undergoes significant biotransformation, with only a small fraction of the unchanged kaempferol able to cross the intestinal barrier [36]. It has been isolated from tea as well as common vegetables and fruits like beans, broccoli, cabbage, grapes, strawberries, tomatoes, apples and grapefruit [37]. Kaempferol has anti-inflammatory and anti-cancer properties, protects the liver and prevent metabolic diseases (Figure 3). The most well-known of its properties are its anti-inflammatory effects by decreasing lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) expression and also by increasing the number of activated macrophages [38]. Kaempferol is a dietary flavonoids that occur in fruits, vegetables, beverages, chocolates, herbs and plants [39] and reported to possess anti-diabetic property.	doab	2025-04-07T03:56:59.112023	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 24 }
0071a6c6-eff7-449d-a548-4af9bff79a91.25	6. Mechanism of antidiabetic action of kaempferol Kaempferol has been reported to lower blood glucose [40], inhibit α -amylase and α -glucosidase [41]. This section addresses the mechanism of action of Kaempferol under the following headings.	doab	2025-04-07T03:56:59.112154	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 25 }
0071a6c6-eff7-449d-a548-4af9bff79a91.26	7. Inhibition of α-amylase and α-glucosidase enzymes. α-Amylase and α-glucosidase are carbohydrate hydrolyzing enzymes located in the digestive tract. α- amylase in the duodenum initiates digestion and catalyzes the hydrolysis of α-1, 4 glycosidic linkages in starch resulting into sugars such as maltose, maltotriose and branched oligosaccharides. Then, α-glucosidase present in the brush border of the intestinal epithelium (enterocytes) is responsible for the final step of carbohydrates digestion, prior to their absorption. This enzyme cleaves 17 Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393 humans [42, 43]. terminal non-reducing 1, 4 linkages and converts the disaccharides and oligosaccharides into glucose, which is then transported by sodium/glucose co-transporter 1 (SGLT1) from the intestinal lumen to the cytosol of enterocytes. In turn, glucose transporter 2 (GLUT2), found in the basolateral membrane of enterocytes, trans- by the inhibition of carbohydrate hydrolyzing enzymes in the digestive tract of kaempferol lowers blood glucose and inhibit α-amylase and α -glucosidase. inhibition of α -amylase and α -glucosidase through kaempferol. sis by targeting the glucose production and metabolic pathways. 8. Maintaining glucose homeostasis 9. Modulation of antioxidant profile 10. Reversal of lipid profile alterations induced diabetic rats [29]. One of the approaches to managing diabetes is to delay the absorption of glucose Controlling the activity of these enzymes slows glucose production in the postprandial stage and this could be a therapeutic approach for people with diabetes. Hence, the search for inhibitors from medicinal plants is a great development [44]. Ibitoye et al. identified that kaempferol from Cucumis sativus L. lowers blood glucose and inhibited the activity of α -amylase and α glucosidase at IC50 of 51.24 and 29.37 μg/mL respectively [29]. This inhibition means reduction in blood glucose in the postprandial stage of alloxan-induced diabetic rats when given 165 mg/kg body weight of kaempferol from Cucumis sativus fruits. This evidently supports that It may be possible that the glucose lowering activity of C. sativus fruits is through Diabetes features dysregulated glucose metabolism characterized by increased hepatic glucose production and decreased glucose oxidation. This eventually leads to deterioration in glucose control. Alkhalidy et al reported that kaempferol ameliorate hyperglycemia and enhance glucose tolerance in insulin deficient mice [45]. Diabetic mice displayed significantly higher pyruvate carboxylase activity. Kaempferol treatment suppressed the elevated pyruvate carboxylase activity and glucose-6 phosphatase activity in the liver suggesting that kaempferol may improve glycemic control in diabetes in part through suppressing gluconeogenesis in the liver via the regulation of pyruvate carboxylase, the first and critical step in gluconeogenesis [45]. It could therefore be a strategy for maintaining glucose homeosta- Generation of reactive oxygen species and free radicals contributes to the pathogenesis of diabetes [46]. This increased ROS production overruns the cellular antioxidant defense system leading to oxidative stress and damage [47]. Some diabetes research confirm this phenomenon in different diabetes model [48, 49]. Catalase, superoxide dismutase and glutathione are reduced significantly in diabetes [50]. Kaempferol reversed the alterations on oxidative stress markers in alloxan- One of the complications in diabetes is dyslipidemia, where the lipid profile is disturbed. It is usually presented with elevated levels of total cholesterol TC, triacylglycerol TAG, and low-density lipoprotein cholesterol LDLc and a reduction of high density lipoprotein cholesterol HDLc [51]. These alterations could predispose to developing ports glucose from cytosol to blood via facilitated diffusion. #### Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393 Cucumber Economic Values and Its Cultivation and Breeding Intestinal permeability study of kaempferol shows it undergoes significant biotransformation, with only a small fraction of the unchanged kaempferol able broccoli, cabbage, grapes, strawberries, tomatoes, apples and grapefruit [37]. Kaempferol has been reported to lower blood glucose [40], inhibit α -amylase and α -glucosidase [41]. This section addresses the mechanism of action of α-Amylase and α-glucosidase are carbohydrate hydrolyzing enzymes located in the digestive tract. α- amylase in the duodenum initiates digestion and catalyzes the hydrolysis of α-1, 4 glycosidic linkages in starch resulting into sugars such as maltose, maltotriose and branched oligosaccharides. Then, α-glucosidase present in the brush border of the intestinal epithelium (enterocytes) is responsible for the final step of carbohydrates digestion, prior to their absorption. This enzyme cleaves It has been isolated from tea as well as common vegetables and fruits like beans, Kaempferol has anti-inflammatory and anti-cancer properties, protects the liver and prevent metabolic diseases (Figure 3). The most well-known of its properties are its anti-inflammatory effects by decreasing lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) expression and also by increasing the number of activated macrophages [38]. Kaempferol is a dietary flavonoids that occur in fruits, vegetables, beverages, chocolates, herbs and plants [39] to cross the intestinal barrier [36]. Figure 3. Biological roles of Kaempferol. and reported to possess anti-diabetic property. Kaempferol under the following headings. 6. Mechanism of antidiabetic action of kaempferol 7. Inhibition of α-amylase and α-glucosidase enzymes. 16 terminal non-reducing 1, 4 linkages and converts the disaccharides and oligosaccharides into glucose, which is then transported by sodium/glucose co-transporter 1 (SGLT1) from the intestinal lumen to the cytosol of enterocytes. In turn, glucose transporter 2 (GLUT2), found in the basolateral membrane of enterocytes, transports glucose from cytosol to blood via facilitated diffusion. One of the approaches to managing diabetes is to delay the absorption of glucose by the inhibition of carbohydrate hydrolyzing enzymes in the digestive tract of humans [42, 43]. Controlling the activity of these enzymes slows glucose production in the postprandial stage and this could be a therapeutic approach for people with diabetes. Hence, the search for inhibitors from medicinal plants is a great development [44]. Ibitoye et al. identified that kaempferol from Cucumis sativus L. lowers blood glucose and inhibited the activity of α -amylase and α glucosidase at IC50 of 51.24 and 29.37 μg/mL respectively [29]. This inhibition means reduction in blood glucose in the postprandial stage of alloxan-induced diabetic rats when given 165 mg/kg body weight of kaempferol from Cucumis sativus fruits. This evidently supports that kaempferol lowers blood glucose and inhibit α-amylase and α -glucosidase. It may be possible that the glucose lowering activity of C. sativus fruits is through inhibition of α -amylase and α -glucosidase through kaempferol.	doab	2025-04-07T03:56:59.112190	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 26 }
0071a6c6-eff7-449d-a548-4af9bff79a91.27	8. Maintaining glucose homeostasis Diabetes features dysregulated glucose metabolism characterized by increased hepatic glucose production and decreased glucose oxidation. This eventually leads to deterioration in glucose control. Alkhalidy et al reported that kaempferol ameliorate hyperglycemia and enhance glucose tolerance in insulin deficient mice [45]. Diabetic mice displayed significantly higher pyruvate carboxylase activity. Kaempferol treatment suppressed the elevated pyruvate carboxylase activity and glucose-6 phosphatase activity in the liver suggesting that kaempferol may improve glycemic control in diabetes in part through suppressing gluconeogenesis in the liver via the regulation of pyruvate carboxylase, the first and critical step in gluconeogenesis [45]. It could therefore be a strategy for maintaining glucose homeostasis by targeting the glucose production and metabolic pathways.	doab	2025-04-07T03:56:59.112505	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 27 }
0071a6c6-eff7-449d-a548-4af9bff79a91.28	9. Modulation of antioxidant profile Generation of reactive oxygen species and free radicals contributes to the pathogenesis of diabetes [46]. This increased ROS production overruns the cellular antioxidant defense system leading to oxidative stress and damage [47]. Some diabetes research confirm this phenomenon in different diabetes model [48, 49]. Catalase, superoxide dismutase and glutathione are reduced significantly in diabetes [50]. Kaempferol reversed the alterations on oxidative stress markers in alloxaninduced diabetic rats [29].	doab	2025-04-07T03:56:59.112539	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 28 }
0071a6c6-eff7-449d-a548-4af9bff79a91.29	10. Reversal of lipid profile alterations One of the complications in diabetes is dyslipidemia, where the lipid profile is disturbed. It is usually presented with elevated levels of total cholesterol TC, triacylglycerol TAG, and low-density lipoprotein cholesterol LDLc and a reduction of high density lipoprotein cholesterol HDLc [51]. These alterations could predispose to developing atherosclerosis and cardiovascular diseases. Reversal of these alterations in alloxandiabetic rats suggests its anti-dyslipidemic capability [29]. Alkhalidy et al. observed that untreated diabetic mice had lower total cholesterol, HDL-cholesterol, and LDLcholesterol levels when compared to non-diabetic mice [45]. Kaempferol treatment reversed these changes to the levels similar to those seen in non-diabetic mice.	doab	2025-04-07T03:56:59.112565	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 29 }
0071a6c6-eff7-449d-a548-4af9bff79a91.30	11. Maintenance of glycoprotein content Glycoproteins are carbohydrate-containing proteins found on the cell membrane. They play important roles in membrane transport, cell differentiation and recognition, adhesion of macromolecules to cell surface and also in the secretion and absorption of macromolecules [52]. Impaired metabolism of glycoproteins contributes to the pathogenesis of diabetes [53]. Studies have reported that alterations in concentrations of various glycoproteins contribute to human diabetes [54]. Elevated levels of glycoproteins in diabetic condition could be a consequence of impaired carbohydrate metabolism [55]. Chandramohan et al. reported that kaempferol reversed elevated level of hexoses, hexosamines, fucose and sialic acid (glycoprotein componets) in streptozotocin-induced diabetic rats which may be due to the activation of glucose transport mechanism and also alters insulin binding receptor specificity [56].	doab	2025-04-07T03:56:59.112615	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 30 }
0071a6c6-eff7-449d-a548-4af9bff79a91.31	Author details Ibitoye Oluwayemisi Beatrice1 \, Ajiboye Taofeek Olakunle2 , Abdulazeez Azeemat Titilola3 , Oyegoke Rukayat Abiodun4 , Muritala Hamdalat Folake4 and Abubakar Fatimah Aluko4 1 Antioxidants, Redox Biology and Toxicology Research Laboratory, Department of Biological Sciences, Al-Hikmah University, Ilorin, Nigeria 2 Antioxidants, Redox Biology and Toxicology Research Group, Department of Medical Biochemistry, College of Health Sciences, Nile University of Nigeria, Abuja, Nigeria 3 Department of Biological Sciences, Al-Hikmah University, Ilorin, Ilorin, Nigeria 4 Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Ilorin, Nigeria \Address all correspondence to: [email protected] © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 19 3577-3581. Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393 [1] Zimmet, P., Alberti, K. G., & Shaw, J. (2001). Global and societal implications of the diabetes epidemic. Nature, 414, [9] Saely, C. H., Aczel, S., Marte, T., Langer, P., & Drexel, H. (2004). Cardiovascular complications in Type 2 diabetes mellitus depend on the coronary angiographic state rather than on the diabetic state. Diabetologia, 47, [10] Wallace, C., Reiber, G. E., LeMaster, J., Smith, D. G., Sullivan, K., Hayes, S., & Vath, C. (2002). Incidence of falls, risk factors for falls, and fallrelated fractures in individuals with diabetes and a prior foot ulcer. Diabetes Care, 25, [11] Ajiboye, T. O., Uwazie, J., Haliru, F., Ibitoye, O., & Sunmonu, T. O. (2017). Mechanisms of action of columbamine, jatrorrhizine and magnoflorine as antidiabetic agents. In J. N. Govil (Ed.), Recent progress in medicinal plant (Vol. 45, pp. 221-232). USA: Studium [12] Antidiabetic drugs. https:// www.amboss.com/us/knowledge/ antidiabetic\_drugs Updated: October 5, 2020. Accessed: November 22, 2020. [13] Marles, R. J., & Farnsworth, N. R. (1995). Antidiabetic plants and their active constituents. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 2: 137-189. [14] Sharma, N., & Garg, V. (2009). Antidiabetic and antioxidant potential of ethanolic extract of Butea monosperma leaves in alloxan-induced diabetic mice. Indian Journal of Biochemistry & Biophysics, 46: 99-105. [15] Sun, H., Fang, W., Wang, W., & Hu, C. (2006). Structure-activity relationships of oleanane- and ursanetype triterpenoids. Botanical Studies, [16] Saidu, A. N., Oibiokpa, F. I. & Olukotun, I. O. (2014). Phytochemical 145-146. 1983-1986. Press LLC. 47: 339-368. Federation Diabetes Atlas Ninth edition [3] American Diabetes Association, The cost of Diabetes https://www.diabetes. org/resources/statistics/cost-diabetes Updated 02/2020. Accessed January [4] Ozcan, U., Cao, Q., Yilmaz, E., Lee, A.-H., Iwakoshi, N. N., Ozdelen, E., & Hotamisligil, G. S. (2004). Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science, [5] Poitout, V., & Robertson, R. P. (2008). Glucolipotoxicity: Fuel excess and cell dysfunction. Endocrine [6] Hove, M. N., Kristensen, J. K., Lauritzen, T., & Bek, T. (2004). The prevalence of retinopathy in an unselected population of type 2 diabetes patients from Arhus County, Denmark. Acta Ophthalmologica Scandinavica, [7] Moran, A., Palmas, W., Field, L., Bhattarai, J., Schwartz, J. E., Weinstock, R. S., & Shea, S. (2004). Cardiovascular autonomic neuropathy is associated with microalbuminuria in older patients with type 2 diabetes. Diabetes Care, 27, [8] Huang, C., Kim, Y., Caramori, M. L., Fish, A. J., Rich, S. S., Miller, M. E., & Mauer, M. (2002). Cellular basis of diabetic nephropathy: II. The transforming growth factor-beta system and diabetic nephropathy lesions in type 1 diabetes. Diabetes, 51, Reviews, 29(3), 351-366. [2] The International Diabetes 2019. Diabetes facts & figures 782-787. References 12, 2021. 306, 457-461 82, 443-448. 972-977. Antidiabetic Principle in Cucumis sativus L. DOI: http://dx.doi.org/10.5772/intechopen.96393	doab	2025-04-07T03:56:59.112686	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 31 }
0071a6c6-eff7-449d-a548-4af9bff79a91.33	Chapter 3 Cucumber Economic Values and Its Cultivation and Breeding [54] Chandramohan, G., Al-Numair, K. S., Alsaif, M. A. & Veeramani, C. (2015). Antidiabetic effect of kaempferol a flavonoid compound, on streptozotocin-induced diabetic rats with special reference to glycoprotein components. Progress in Nutrition, Vol. [55] Peppa, M., Stavroulakis, P. & Raptis, S. A. (2009). Advanced glycoxidation products and impaired diabetic wound healing. Wound Repair [56] Marshall, S., Bacote, V. & Traxinger, R. R. (1991). Discovery of metabolic pathway mediating glucose induced desensitization of glucose transport system: role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem 1991; 266: 4706-4712. Regeneration, 17; 461-472. 17, N. 1: 50-57. and diabetes. Free Radical Biology & [47] Sies, H. (1991). Oxidative stress: From basic research to clinical application. The American Journal of [48] Oloyede, H. O. B., Ajiboye, T. O., Abdussalam, A. F., &. Adeleye, A. O. (2014). Blighia sapida leaves halt elevated blood glucose, dyslipidemia and oxidative stress in alloxaninduced diabetic rats. Journal of Ethnopharmacology, 157, 309-319. [49] Oloyede, H. O. B., Bello, T. O., Ajiboye, T. O., & Salawu, M. O. (2015). Antidiabetic and antidyslipidemic activities of aqueous leaf extract of Dioscoreophyllum cumminsii (Stapf) Diels in alloxan-induced diabetic rats. Journal of Ethnopharmacology, 166, [50] Ajiboye, T. O., Akinpelu, S. A., Muritala, H. F., Ogunbode, S. M., Adeleye, A. O., Oladiji, A. T., & Oloyede, O. B. (2014). Trichosanthes dyslipidemia, protein oxidation, lipid peroxidation and DNA fragmentation in the liver of high-fat diet-fed rats. Journal of Food Biochemistry, 38, 480-490. [51] Mittal, N., Keaur, J. & Mahmood, A. (1996). Changes in tubular membrane glycosylation in diabetic insulin and thyroxin treated rat kidneys. Indian Journal of Experimental Biology, 34: [52] Knecht KT, Bradford BU, Mason RP & Thurman RG. (1990). In vivo formation of free radicals metabolite of ethanol. Molecular Pharmacology, [53] Sharma, C., Dalferes, F. R., diabetic rats. Biochem Int, 1987; Radhakrishnamurthy, B., De-Paolo, C. J. & Berenson, G. S. (1987). Hepatic glycoprotein synthesis in streptozotocin cucumerina fruit extenuates Medicine, 50, 567-575. Medicine, 91, 31S–38S. 313-322. 782-785. 38: 26-30. 36: 15-19. 22	doab	2025-04-07T03:56:59.112953	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 33 }
0071a6c6-eff7-449d-a548-4af9bff79a91.34	Cucumber Pickles and Fermentations Sarmad Ghazi Al-Shawi and Sadiq Jaafir Aziz Alneamah	doab	2025-04-07T03:56:59.113107	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 34 }
0071a6c6-eff7-449d-a548-4af9bff79a91.35	Abstract Cucumber sometimes used in sodium chloride solution as a substrate in lactic acid bacteria fermentation. The good fermentation always depending on many overlapped physical, chemical and microbial factors related with suspension of a strong and porous vegetables in a fluid. Keeping the cucumber integrity is very critical issue, and this may affect on the fermentation of liquid ingredients. This chapter tries to focus on the current efforts that conducting to control on the factors that affecting on cucumber fermentation. Modern and advanced technologies of recent studies are included within this chapter just like reducing the brining sodium chloride concentration, fresh cucumber gas exchange to develop their brining properties by using lactic acid bacteria cultures, developing an anaerobic cucumber fermentation tank system; preventing of cucumber gaseous spoilage by pouring of CO2 from fermentation brines. Keywords: lactic acid bacteria, cucumbers, fermentation, sodium chloride, brine ## 1. Introduction #### 1.1 Historical perspective Different fermented foods could be categorized according to fermentation products just like organic acids which consisting of acetic acid and lactic acid (dairy and vegetables); and peptides and amino acids resulted from protein (fish and other fermentations); CO2 (bread); and alcohol (wine and beer) [1, 2]. Food fermentation is one of an early the most precise innovations created and developed by people. In Asia, coastal foragers during the age of primitive pottery (8000 to 3000 b.c.) were thought to have fermented vegetables before developing of crop-based agriculture [3]. It is possible that dairy fermentations in Middle East came after cattle domestication, alcohol was the first discovered fermented product from fruit fermentation. Many advanced fermentation procedures to produce alcohol by using the cereals were created nearly 4000 b.c., just like producing wine from rice in Asia and beer in Egypt [1]. In Asia, many composed references regarding fermentation innovation were found in historic poems Shijing Chinese book (1100 to 600 b.c.), that celebrates "the thousand wines of Yao," in referring to a kingdom in China from 2300 b.c. Cucumber thought were first fermented nearly 2000b.c. in Middle East. Old composed records came from the remains of papers of a play (The Taxiarchs) by Eupolis a writer from Greece (429–412 b.c.), also in Christian Bible, pickles were repeated many times. The fermented cabbage and kimchi on the Korean style, is expected to have established in the primitive pottery age from the wizened vegetables ordinary fermentation stored in seawater [1]. Sauerkraut on European style is thought was established in China, while the technique might be transferred to Europe at the invasion time of Mongol to central Europe in the 13th century. Nowadays, the vegetable fermentation industry is conducting on an enormous scale. Companies in United States that working on cucumber pickles fermentations may have 1000 fermentation tanks of fortythousand-liter capacity at one site. Throughout the ages, it was believed that cucumber pickle as the fairly fermented cucumber to which spices, vinegar, salt, and sometimes sugar has been added. While the preservation was not required by using the heat. Recently, fresh packed pickles, manufactured by adding of spices, salt, and vinegar to the fresh cucumbers under pasteurized preservation, are representing a huge portion of pickle industry. Industrial treatments tentatively preserve around 40% of crop through the fermentation in NaCI brines that contain fermentable carbohydrates which converting into acetic acids, ethanol, lactic acid, CO2, and other compounds by naturally existence lactic acid bacteria and yeast. This procedure uses to expand the using equipment packing line and workers to throughout the year operation in manufacturing of the final product. Traditionally, fiberglass, wood, and polyethylene tanks are used for the fermentation that might require 10–21 days (period of storage in the same tanks is generally less than 1 year) and sometimes longer. Tanks are put outdoors to give the opportunity for sunlight ultraviolet irradiation to hit the surface of the brine and subsequently inhibiting yeasts and molds growing, and other microorganisms on the surface of the brine. During the fermentation of cucumber pickles, brine storage and processing operations are liable to the reactions of oxidation which affect adversely on the quality properties. In spite of pickles are flooded in brine during fermentation and bulk-storage, while the containers are opening, which encourage the exposure to air and sunlight. Additionally, pickle tanks' brines are usually spread with air in order to mix the components and to release CO2, and at the time of transferring to processing operations, pickles are removed from brine and subsequently exposes to light and oxygen. Also, the brines and pickles content of traces prooxidant metals just like copper, zinc and iron which act together with oxygen and light to be in charge of pigments oxidation and developing undesirable flavors sometimes, and this may lead to considerable economic loss of the market value. #### 1.2 Cucumber fermentations Cucumber (Cucumis sativus) fermentation in United States is conducted in 30,000–40,000 liter, fiberglass tanks with open top and placed outdoors to allowing the surface to exposure to sunlight. Sunlight UV radiation is dependent to suppression the surface aerobic yeasts that have the ability to utilize lactic acid that resulted from fermentation. Cucumbers are submerged totally with salt brine and kept under the brine surface with wooden headboards. Fermentation is usually conducted in 6% NaCl. Calcium chloride typically added the cover brine in order to keep the fragile texture, and firm of the fermented cucumber throughout fermentation and storage [4]. The fermentations of cucumber usually subject a homolactic acid fermentation, that is not resulting CO2 from sugars. Although CO2 could be produced via cucumbers respiration and via malate decarboxylation over the beginning of fermentation [4]. Some of lactic acid bacteria have an analytical malolactic 25 Cucumber Pickles and Fermentations processing [7]. DOI: http://dx.doi.org/10.5772/intechopen.96052 enzyme that converting malate to lactate and CO2. The reaction of malolactic enzyme takes place intracellularly resulting in proton absorption, subsequently increasing the internal pH of the cell. Although it is a recommendable reaction in winemaking (applied to removing the acidity of wines), the fermentation of malolactic in cucumbers may lead to formation of "bloaters," or undesirable pockets of internal gas, resulting in decreasing the yield of the production [5]. In order to prevent the formation of bloater, the fermentation of cucumber is clean with air to get rid the surplus CO2 from the tank [6]. In order to restrict the growing of aerobic microorganisms in air-purged cucumber fermentations, especially molds and yeasts, acetic acid (0.16%) or potassium sorbate (~0.04%) could be used as aids in Air purging may be stopped each day several hours to control aerobic microorganisms' excessive growth. Usually, cucumber is fermented with Lactobacillus. plantarum and other LAB and may store for year in fermentation tanks in degrees under than 0 °C while NaCl concentration commonly increase to 10–15% during the storage to reduce freezing damage and keeping the required fermented cucumber texture. Cucumber should be washed before selling in order to remove the excess salt and then using different packages (jars, pouches, plastic pails) with suitable covers in packaging. The covers usually contain spices, acetic acid, and lactic acid residues. Pasteurization sometimes is used for fermented pickles while heat treatment is not used for big containers. Excessive growth of microorganisms is eliminated by low pH, organic acids, and absence of fermentable sugars. Cucumber fermentations depend on the growing of LAB that existence naturally on cucumbers surface. Although, some starter cultures are added to cucumber fermentation to get a consistent product, adding Lactobacillus plantarum does not decarboxylate malic acid (subsequently does not form bloaters) [8], and this approach has been created, developed, and tasted to identify culture growing capability in cucumber fermentations [9]. A procedure for starter culture preparation that suitable for the requirements of kosher is applicable to producers [10]. The brined cucumbers' primary pH is nearly 6.5. Recycled brine could be used in commercial fermentations, or adding acetic acid to brine solutions. This acid addition may help in removing the excess CO2 and encouraging LAB growth, so the commercial fermentations' primary pH could vary basically. Some of the metabolites could have an inhibitory effect on the other bacteria just like peroxides, bacteriocins, and peptides [11]. There might be 1.5% lactic acid, pH (3.1–3.5), few or no sugar at the end of fermentation. In such an environment that is acidic, anaerobic, high salty, and lacks sugar, there are a low number of microorganisms that have the ability to grow and survive to preserve cucumbers. Sometimes during storage, fermented cucumbers expose secondary undesired fermentation which is identified by pH increasing, lactic acid vanishing, propionic and butyric acid formation. Deterioration of fermented cucumber happening at the spring season beginning when increasing the surrounding temperature. Increasing propionic and butyric acid concentrations lead to smelly spoilage [12]. The microbial environment of this spoilage presently is not closely defined but may attribute to the growth of bacteria that form spores such as clostridia when increasing the pH above 4.6. The salt concentration of the fermented cucumbers is about (6% or more) and this is very high for consumption directly by humans. Therefore, the salt concentration is reduced to around 2% by water washing directly before packing and distribution. This treatment lead to high salt concentrations of the waste stream in addition to a high BOD resulting from the organic ingredients that are existed in the brine and that spread out of cucumbers over the process of desalting. Hence, cucumber brine of the desalting process is commonly recycled and might be utilized another fermentation [13]. The brines fermentation could be treated in order to expel the softening enzymes (mostly polygalacturonases) before #### Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Economic Values and Its Cultivation and Breeding etables ordinary fermentation stored in seawater [1]. thousand-liter capacity at one site. pickle industry. turing of the final product. the surface of the brine. 1.2 Cucumber fermentations and sunlight. expected to have established in the primitive pottery age from the wizened veg- Sauerkraut on European style is thought was established in China, while the technique might be transferred to Europe at the invasion time of Mongol to central Europe in the 13th century. Nowadays, the vegetable fermentation industry is conducting on an enormous scale. Companies in United States that working on cucumber pickles fermentations may have 1000 fermentation tanks of forty- Throughout the ages, it was believed that cucumber pickle as the fairly fermented cucumber to which spices, vinegar, salt, and sometimes sugar has been added. While the preservation was not required by using the heat. Recently, fresh packed pickles, manufactured by adding of spices, salt, and vinegar to the fresh cucumbers under pasteurized preservation, are representing a huge portion of Industrial treatments tentatively preserve around 40% of crop through the fermentation in NaCI brines that contain fermentable carbohydrates which converting into acetic acids, ethanol, lactic acid, CO2, and other compounds by naturally existence lactic acid bacteria and yeast. This procedure uses to expand the using equipment packing line and workers to throughout the year operation in manufac- Traditionally, fiberglass, wood, and polyethylene tanks are used for the fermentation that might require 10–21 days (period of storage in the same tanks is generally less than 1 year) and sometimes longer. Tanks are put outdoors to give the opportunity for sunlight ultraviolet irradiation to hit the surface of the brine and subsequently inhibiting yeasts and molds growing, and other microorganisms on During the fermentation of cucumber pickles, brine storage and processing operations are liable to the reactions of oxidation which affect adversely on the quality properties. In spite of pickles are flooded in brine during fermentation and bulk-storage, while the containers are opening, which encourage the exposure to air Additionally, pickle tanks' brines are usually spread with air in order to mix the components and to release CO2, and at the time of transferring to processing operations, pickles are removed from brine and subsequently exposes to light and oxygen. Also, the brines and pickles content of traces prooxidant metals just like copper, zinc and iron which act together with oxygen and light to be in charge of pigments oxidation and developing undesirable flavors sometimes, and this may Cucumber (Cucumis sativus) fermentation in United States is conducted in 30,000–40,000 liter, fiberglass tanks with open top and placed outdoors to allowing the surface to exposure to sunlight. Sunlight UV radiation is dependent to suppression the surface aerobic yeasts that have the ability to utilize lactic acid that resulted from fermentation. Cucumbers are submerged totally with salt brine and kept under the brine surface with wooden headboards. Fermentation is usually conducted in 6% NaCl. Calcium chloride typically added the cover brine in order to keep the fragile texture, and firm of the fermented cucumber throughout fermentation and storage [4]. The fermentations of cucumber usually subject a homolactic acid fermentation, that is not resulting CO2 from sugars. Although CO2 could be produced via cucumbers respiration and via malate decarboxylation over the beginning of fermentation [4]. Some of lactic acid bacteria have an analytical malolactic lead to considerable economic loss of the market value. 24 enzyme that converting malate to lactate and CO2. The reaction of malolactic enzyme takes place intracellularly resulting in proton absorption, subsequently increasing the internal pH of the cell. Although it is a recommendable reaction in winemaking (applied to removing the acidity of wines), the fermentation of malolactic in cucumbers may lead to formation of "bloaters," or undesirable pockets of internal gas, resulting in decreasing the yield of the production [5]. In order to prevent the formation of bloater, the fermentation of cucumber is clean with air to get rid the surplus CO2 from the tank [6]. In order to restrict the growing of aerobic microorganisms in air-purged cucumber fermentations, especially molds and yeasts, acetic acid (0.16%) or potassium sorbate (~0.04%) could be used as aids in processing [7]. Air purging may be stopped each day several hours to control aerobic microorganisms' excessive growth. Usually, cucumber is fermented with Lactobacillus. plantarum and other LAB and may store for year in fermentation tanks in degrees under than 0 °C while NaCl concentration commonly increase to 10–15% during the storage to reduce freezing damage and keeping the required fermented cucumber texture. Cucumber should be washed before selling in order to remove the excess salt and then using different packages (jars, pouches, plastic pails) with suitable covers in packaging. The covers usually contain spices, acetic acid, and lactic acid residues. Pasteurization sometimes is used for fermented pickles while heat treatment is not used for big containers. Excessive growth of microorganisms is eliminated by low pH, organic acids, and absence of fermentable sugars. Cucumber fermentations depend on the growing of LAB that existence naturally on cucumbers surface. Although, some starter cultures are added to cucumber fermentation to get a consistent product, adding Lactobacillus plantarum does not decarboxylate malic acid (subsequently does not form bloaters) [8], and this approach has been created, developed, and tasted to identify culture growing capability in cucumber fermentations [9]. A procedure for starter culture preparation that suitable for the requirements of kosher is applicable to producers [10]. The brined cucumbers' primary pH is nearly 6.5. Recycled brine could be used in commercial fermentations, or adding acetic acid to brine solutions. This acid addition may help in removing the excess CO2 and encouraging LAB growth, so the commercial fermentations' primary pH could vary basically. Some of the metabolites could have an inhibitory effect on the other bacteria just like peroxides, bacteriocins, and peptides [11]. There might be 1.5% lactic acid, pH (3.1–3.5), few or no sugar at the end of fermentation. In such an environment that is acidic, anaerobic, high salty, and lacks sugar, there are a low number of microorganisms that have the ability to grow and survive to preserve cucumbers. Sometimes during storage, fermented cucumbers expose secondary undesired fermentation which is identified by pH increasing, lactic acid vanishing, propionic and butyric acid formation. Deterioration of fermented cucumber happening at the spring season beginning when increasing the surrounding temperature. Increasing propionic and butyric acid concentrations lead to smelly spoilage [12]. The microbial environment of this spoilage presently is not closely defined but may attribute to the growth of bacteria that form spores such as clostridia when increasing the pH above 4.6. The salt concentration of the fermented cucumbers is about (6% or more) and this is very high for consumption directly by humans. Therefore, the salt concentration is reduced to around 2% by water washing directly before packing and distribution. This treatment lead to high salt concentrations of the waste stream in addition to a high BOD resulting from the organic ingredients that are existed in the brine and that spread out of cucumbers over the process of desalting. Hence, cucumber brine of the desalting process is commonly recycled and might be utilized another fermentation [13]. The brines fermentation could be treated in order to expel the softening enzymes (mostly polygalacturonases) before the recycling [14], which acts on degrading cucumber cell's pectic substances and softening the fruits. #### 1.3 Critical factors for fermenting cucumbers Fermentation is influenced by variables due to cucumbers, environmental conditions under which they are kept during fermentation, and microorganisms that are naturally present or intentionally added. Since it is so important to maintain the structural integrity of cucumbers, both physical and chemical factors are involved. The interactions between these factors lead to an extremely interesting and complex fermentation process [15]. A lot of research on the fermentation of cucumbers and other fruits and vegetables has been done. However, there is an incomplete understanding of the interactions between the microbiological, chemical, and physical factors involved. Before the cucumber fermentation industry can take full advantage of the biotechnology revolution that looms for many fermentation industries, more understanding of these interactions is needed [16]. #### 1.4 Microbial changes in spoilage The production of CO2 in the cover brine of fermenting vegetables by heterofermentative LAB and fermentative species of yeasts has been linked with gas pockets formation inside the cucumber, which called formation of bloater (Figure 1). Homofermentative LAB capable of decarboxylating malic acid, as example L. plantarum, might cause bloating by producing a sufficient CO2 when combined with the CO2 formed from the respiring vegetable tissues [8, 18]. Prevention of bloater formation was effective in fermented cucumber brines by using nitrogen or air [6, 19]. Air purging has to be carefully controlled as it may result in fruit softening due to mold growth [20, 21] reduced brine acidity due to yeast growth and off-colors and flavors. The addition of potassium sorbate to fermentation brines, including the application of spray to brine surfaces, is widely used to minimize the growth of yeast and the development of CO2. Oxidative yeasts may cause malodorous spoilage of fermented cucumbers to develop. The lactic acid generated during fermentation can be consumed by these microorganisms, with a subsequent increase in pH that facilitates the development of spoilage microorganisms [22, 23]. In cucumbers, lactic acid produced during primary fermentation can be catabolized by yeasts of the genera Pichia and Issatchenkia, causing an increase in pH. Pectinolytic enzymes derived from plant material or microbes can cause the softening of brined vegetables (Figure 2). Mold growth accompanying film-forming yeast growth on the brine surface can cause softening of cucumbers. In the absence of sunlight and the presence of minimal amounts of oxygen, heavy scum yeast and/or mold growth is generally the result of neglecting brine material during extended storage [25]. In order to maintain anaerobic conditions and to limit the growth of surface yeasts and molds, Pickled cucumber tanks are usually held indoors, with a seated plastic cover weighted down with water or brine. Mold polygalacturonases associated with cucumber flowers can also result in the softening of brined cucumbers [26]. By draining and rebrining the tank with calcium chloride, this problem can be reduced. 36 hours after the initial brining procedure. However, this solution is not about salt disposal. Recycled brines are instead treated to inactivate the softening enzymes, if necessary [14]. The addition of calcium chloride may slow down the rate of fermenting cucumbers' enzymatic softening. This should not, however, be relied upon 27 Figure 1. Steps brine fermentation of cucumber [17]. to eliminate problems with enzymatic softening. Care must be taken to minimize the contamination of flowers and plant debris by cucumbers, especially small fruits, which may be a source of contamination by pectinolytic molds. Due to the reduced Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Economic Values and Its Cultivation and Breeding 1.3 Critical factors for fermenting cucumbers standing of these interactions is needed [16]. growth of yeast and the development of CO2. Issatchenkia, causing an increase in pH. softening of brined vegetables (Figure 2). 1.4 Microbial changes in spoilage softening the fruits. factors involved. the recycling [14], which acts on degrading cucumber cell's pectic substances and Fermentation is influenced by variables due to cucumbers, environmental conditions under which they are kept during fermentation, and microorganisms that are naturally present or intentionally added. Since it is so important to maintain the structural integrity of cucumbers, both physical and chemical factors are involved. The interactions between these factors lead to an extremely interesting and complex fermentation process [15]. A lot of research on the fermentation of cucumbers and other fruits and vegetables has been done. However, there is an incomplete understanding of the interactions between the microbiological, chemical, and physical Before the cucumber fermentation industry can take full advantage of the biotechnology revolution that looms for many fermentation industries, more under- The production of CO2 in the cover brine of fermenting vegetables by heterofermentative LAB and fermentative species of yeasts has been linked with gas pockets formation inside the cucumber, which called formation of bloater (Figure 1). Homofermentative LAB capable of decarboxylating malic acid, as example L. plantarum, might cause bloating by producing a sufficient CO2 when combined with the CO2 formed from the respiring vegetable tissues [8, 18]. Prevention of bloater formation was effective in fermented cucumber brines by using nitrogen or air [6, 19]. Air purging has to be carefully controlled as it may result in fruit softening due to mold growth [20, 21] reduced brine acidity due to yeast growth and off-colors and flavors. The addition of potassium sorbate to fermentation brines, including the application of spray to brine surfaces, is widely used to minimize the Oxidative yeasts may cause malodorous spoilage of fermented cucumbers to develop. The lactic acid generated during fermentation can be consumed by these microorganisms, with a subsequent increase in pH that facilitates the development of spoilage microorganisms [22, 23]. In cucumbers, lactic acid produced during primary fermentation can be catabolized by yeasts of the genera Pichia and Pectinolytic enzymes derived from plant material or microbes can cause the Mold growth accompanying film-forming yeast growth on the brine surface can cause softening of cucumbers. In the absence of sunlight and the presence of minimal amounts of oxygen, heavy scum yeast and/or mold growth is generally the result of neglecting brine material during extended storage [25]. In order to maintain anaerobic conditions and to limit the growth of surface yeasts and molds, Pickled cucumber tanks are usually held indoors, with a seated plastic cover weighted down with water or brine. Mold polygalacturonases associated with cucumber flowers can also result in the softening of brined cucumbers [26]. By draining and rebrining the tank with calcium chloride, this problem can be reduced. 36 hours after the initial brining procedure. However, this solution is not about salt disposal. Recycled brines are instead treated to inactivate the softening enzymes, if necessary [14]. The addition of calcium chloride may slow down the rate of fermenting cucumbers' enzymatic softening. This should not, however, be relied upon 26 to eliminate problems with enzymatic softening. Care must be taken to minimize the contamination of flowers and plant debris by cucumbers, especially small fruits, which may be a source of contamination by pectinolytic molds. Due to the reduced Figure 2. Lactobacillus plantarum cells colonizing the cucumber tissue [24]. amount of brine surface in contact with air compared to the total volume, softening is not a very serious problem in bulk Spanish-style cucumber fermentation. Yeasts and/or molds on the plastic drums used during the conditioning operations (sizing, grading, pitting, stuffing, etc.) can, however, cause softening [22]. Desalting is used to prepare non-pasteurized fermented cucumbers, followed by the addition of cover liquor, often containing acetic acid and preservatives. Sugar is added to sweet pickles at concentrations of up to 40 percent. The main spoilage organisms in such products are osmotolerant yeasts, and a preservation prediction chart, based on the concentration of acid and sugar required for shelf stability, has been developed. On the surface of the liquid, aerobic molds and film yeasts may grow, mainly as a result of defective jar closure. Spoilage microorganisms in sweet pickles include yeasts [27] and lactobacilli, particularly the heterofermentative Lactobacillus fructivorans. In order to prevent the growth of LAB and yeast, non-fermented pickle products in which acetic acid is added to fresh cucumbers (known as fresh-pack pickles) are pasteurized. Recommended procedures include 165 °F (74 °C) for 15 minutes, as described by [28]. Spoilage usually occurs due to improper processing (insufficient heat to pasteurize) and/or improper acidification of pasteurized pickle products, so that a balanced brine product of pH 3.8 to 4.0 is not achieved. Molds and film yeasts are factors in cases of poor jar closure, where oxygen is introduced into the container, as with sweet pickles. This can lead to a potentially dangerous situation triggered by an increase in pH as the spoilage microorganisms consume organic acids. Germination of Clostridium botulinum spores can occur if the pH rises above 4.6. Non-acidified refrigerated products are sold commercially under a variety of names, including half-sour dills, real kosher dills, new kosher dills, sour overnight dills, garlic pickles, new half-sour pickles, new half-sour pickles, new half-sours, new home-style pickles, etc. [29]. These cucumbers may be kept at room temperature in barrels for a few days or longer and then refrigerated at 2–5 ° C to allow fermentation to occur. Microbial growth, enzymatic activity, and the curing process continue at a slow rate under cooling conditions [29]. The gaseous spoilage of the product is caused primarily by the previously mentioned microbial groups that form gas. Due to the much lower concentrations of salt added to these product types, softening issues in refrigeratedfermented products may develop. To such products, fresh, whole garlic cloves and 29 Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 ment of natural fermentation with lactic acid. tives such as potassium sorbate or sodium benzoate [31]. Usually, fermentation is defined as an anaerobic process. Within the cucumber fermentation process, LAB and yeast convert glucose and fructose into lactic acid, ethanol, acetic acid, and CO2. The homofermentative LAB main pathway is breaking down of one six-carbon sugar (glucose) to produce two molecules of three-carbon lactic acid. More complex metabolism is used by Heterofermentative organisms. At the beginning, glucose is converted into CO2 five-carbon sugar phosphate, and furthermore degraded into lactic acid and a two-carbon compound, acetic acid or ethanol [32]. We shall concentrate here on vegetable fermentation biochemical features that link to quality of the product. So far, many researches are paying more attention in vegetables fermentation and storage, especially cucumbers, with reduced salt. Vegetable fermentations' chloride waste can be extremely reduced in case of reducing the required salt for fermentation and storage in order to exclude the desalting step before the conversion to final products. Many research studied the relationship between concentration and type of the salt [33]. Replacing 1.5 Fermentations biochemistry other spices are normally added. It is possible that these spices contain softening enzymes. Whether the half-sour products are manufactured in bulk or in the retail jar, for more than a few weeks, the very nature of the product makes it difficult to maintain good quality. The barreled product achieves the Good Manufacturing Practices (GMP)-recommended brine pH of 4.6 or lowers for acidified foods typically before or shortly after refrigeration, and then slowly begins to produce acid. For a product made in a retail jar, this recommended condition for brine-product pH cannot be ensured because there is no uniform process adopted by the packers in which the product is initially acidified or intentionally incubated for the develop- The refrigerated fresh-pack (non-fermented) products contain 2–3 percent NaCl and sometimes sodium benzoate or other preservatives and are acidified with vinegar at a balanced pH of around 3.7 [29]. The cucumbers are not heated, like the half-sour pickles, either before or after packing. The products will maintain an acceptable quality for several months if properly acidified, refrigerated, and preserved. However, recipes containing no vinegar or other acid in the initial cover liquor should be considered with caution. Quality assurance of cucumber products begins with the removal of the cucumber's outer leaves and woody core. In addition to its undesirable texture, the existing sucrose in the core could be utilized by Lactobacillus mesenteroides resulting in formation of dextran which lead to a stringy and slimy texture. Cucumbers marketed under refrigerated conditions are preserved by the addition of sodium benzoate and metabisulfite [30]. Chemical changes that can result in discoloration (browning) and the formation of objectionable flavors influence the shelf life of such products. The growth of naturally occurring yeasts in cucumbers may result from uneven salting during cucumber preparation and may induce pink coloring and vegetable softening. Spanish-style olives were formerly preserved in cover solutions containing relatively high salt concentrations through fermentation. However, it has been demonstrated that an appropriate combination of low pH (3.5), combined acidity (0.025) mill equivalents (mEq)/L) and moderate proportions of acid (>20.4%) and salt (>25.0%) is also able to preserve well-cured cucumbers [31]. Incompletely cured cucumbers or those with characteristics outside the ranges necessary for complete stabilization without heat treatment have been gradually used to allow pasteurization to be commercialized [22]. In some cases, particularly when pasteurization is not recommended (plastic bags, seasoned olives, etc.), producers used authorized preserva- #### Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Economic Values and Its Cultivation and Breeding Lactobacillus plantarum cells colonizing the cucumber tissue [24]. amount of brine surface in contact with air compared to the total volume, softening is not a very serious problem in bulk Spanish-style cucumber fermentation. Yeasts and/or molds on the plastic drums used during the conditioning operations (sizing, grading, pitting, stuffing, etc.) can, however, cause softening [22]. Desalting is used to prepare non-pasteurized fermented cucumbers, followed by the addition of cover liquor, often containing acetic acid and preservatives. Sugar is added to sweet pickles at concentrations of up to 40 percent. The main spoilage organisms in such products are osmotolerant yeasts, and a preservation prediction chart, based on the concentration of acid and sugar required for shelf stability, has been developed. On the surface of the liquid, aerobic molds and film yeasts may grow, mainly as a result of defective jar closure. Spoilage microorganisms in sweet pickles include yeasts [27] and lactobacilli, particularly the heterofermentative Lactobacillus fructivorans. In order to prevent the growth of LAB and yeast, non-fermented pickle products in which acetic acid is added to fresh cucumbers (known as fresh-pack pickles) are pasteurized. Recommended procedures include 165 °F (74 °C) for 15 minutes, as described by [28]. Spoilage usually occurs due to improper processing (insufficient heat to pasteurize) and/or improper acidification of pasteurized pickle products, so that a balanced brine product of pH 3.8 to 4.0 is not achieved. Molds and film yeasts are factors in cases of poor jar closure, where oxygen is introduced into the This can lead to a potentially dangerous situation triggered by an increase in pH as the spoilage microorganisms consume organic acids. Germination of Clostridium botulinum spores can occur if the pH rises above 4.6. Non-acidified refrigerated products are sold commercially under a variety of names, including half-sour dills, real kosher dills, new kosher dills, sour overnight dills, garlic pickles, new half-sour pickles, new half-sour pickles, new half-sours, new home-style pickles, etc. [29]. These cucumbers may be kept at room temperature in barrels for a few days or longer and then refrigerated at 2–5 ° C to allow fermentation to occur. Microbial growth, enzymatic activity, and the curing process continue at a slow rate under cooling conditions [29]. The gaseous spoilage of the product is caused primarily by the previously mentioned microbial groups that form gas. Due to the much lower concentrations of salt added to these product types, softening issues in refrigeratedfermented products may develop. To such products, fresh, whole garlic cloves and 28 Figure 2. container, as with sweet pickles. other spices are normally added. It is possible that these spices contain softening enzymes. Whether the half-sour products are manufactured in bulk or in the retail jar, for more than a few weeks, the very nature of the product makes it difficult to maintain good quality. The barreled product achieves the Good Manufacturing Practices (GMP)-recommended brine pH of 4.6 or lowers for acidified foods typically before or shortly after refrigeration, and then slowly begins to produce acid. For a product made in a retail jar, this recommended condition for brine-product pH cannot be ensured because there is no uniform process adopted by the packers in which the product is initially acidified or intentionally incubated for the development of natural fermentation with lactic acid. The refrigerated fresh-pack (non-fermented) products contain 2–3 percent NaCl and sometimes sodium benzoate or other preservatives and are acidified with vinegar at a balanced pH of around 3.7 [29]. The cucumbers are not heated, like the half-sour pickles, either before or after packing. The products will maintain an acceptable quality for several months if properly acidified, refrigerated, and preserved. However, recipes containing no vinegar or other acid in the initial cover liquor should be considered with caution. Quality assurance of cucumber products begins with the removal of the cucumber's outer leaves and woody core. In addition to its undesirable texture, the existing sucrose in the core could be utilized by Lactobacillus mesenteroides resulting in formation of dextran which lead to a stringy and slimy texture. Cucumbers marketed under refrigerated conditions are preserved by the addition of sodium benzoate and metabisulfite [30]. Chemical changes that can result in discoloration (browning) and the formation of objectionable flavors influence the shelf life of such products. The growth of naturally occurring yeasts in cucumbers may result from uneven salting during cucumber preparation and may induce pink coloring and vegetable softening. Spanish-style olives were formerly preserved in cover solutions containing relatively high salt concentrations through fermentation. However, it has been demonstrated that an appropriate combination of low pH (3.5), combined acidity (0.025) mill equivalents (mEq)/L) and moderate proportions of acid (>20.4%) and salt (>25.0%) is also able to preserve well-cured cucumbers [31]. Incompletely cured cucumbers or those with characteristics outside the ranges necessary for complete stabilization without heat treatment have been gradually used to allow pasteurization to be commercialized [22]. In some cases, particularly when pasteurization is not recommended (plastic bags, seasoned olives, etc.), producers used authorized preservatives such as potassium sorbate or sodium benzoate [31].	doab	2025-04-07T03:56:59.113139	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 35 }
0071a6c6-eff7-449d-a548-4af9bff79a91.36	1.5 Fermentations biochemistry Usually, fermentation is defined as an anaerobic process. Within the cucumber fermentation process, LAB and yeast convert glucose and fructose into lactic acid, ethanol, acetic acid, and CO2. The homofermentative LAB main pathway is breaking down of one six-carbon sugar (glucose) to produce two molecules of three-carbon lactic acid. More complex metabolism is used by Heterofermentative organisms. At the beginning, glucose is converted into CO2 five-carbon sugar phosphate, and furthermore degraded into lactic acid and a two-carbon compound, acetic acid or ethanol [32]. We shall concentrate here on vegetable fermentation biochemical features that link to quality of the product. So far, many researches are paying more attention in vegetables fermentation and storage, especially cucumbers, with reduced salt. Vegetable fermentations' chloride waste can be extremely reduced in case of reducing the required salt for fermentation and storage in order to exclude the desalting step before the conversion to final products. Many research studied the relationship between concentration and type of the salt [33]. Replacing of NaCl with various cations and anions on fermentation of sugar in cucumber juice. The most interesting thing, fructose was the most preferred fermentable sugar to Lactobacillus plantarum more than glucose in most of experiments. Along with addition of different salts, the utilization of sugar was decreasing as anion or cation concentrations increasing. Lu et al. [33] and Zhou et al. [34] have identified various volatile ingredients in cucumbers that fermented with Lactobacillus plantarum (2% NaCl). About 37 volatile ingredients were determined, and as a result of fermentation, there was a little change in most of them. Inhibition of (E, Z)-2,6 nonadienal and 2-nonenal production was the most outstanding fermentation effect on cucumber volatiles [35]. Characterized trans- and cis-4-hexenoic acid as the strongest odors that specify the brine aroma properties of commercially fermented cucumbers in nearly 6% NaCl [36]. Illustrated that exposing the slurries (2% NaCl) of fermented cucumber to oxygen resulting in formation of nonenzymatic hexanal plus a series of trans unsaturated aldehydes with 5–8 carbon atoms that linked with oxidized odor intensity development the tissue of fermented cucumber. In the existence of light, about 100 μg/ml concentration of calcium disodium EDTA preserve nonfermented pickles against bleaching of pigments and lipid oxidation [14]. Although, when using this compound, there was a little reduction in pickles' firmness retention. Firmness retention in cucumbers fermentation and storage is a key quality issue. It is difficult to assure the firmness retention (in reduced salt fermented cucumbers) equal to what can be accomplished by fermenting and storage in 6% NaCl or more. Nevertheless, over many previous years there was a wide understanding for softening of cucumber tissue. Fleming et al. [21] showed the importance of calcium in keeping fermented cucumbers' firmness. It was found that first-order kinetics is followed by the nonenzymatic softening of acidified, blanched cucumber tissue [37]. The mentioned kinetic manner made it reasonable to identify the activation of entropy and enthalpy of cucumbers' nonenzymatic softening, although that the chemical reactions in charge of softening were not known. At 1.5 M NaCl, both activation of entropy and enthalpy were high. Cucumber softening was inhibited by calcium because it reduced activation entropy too much into a limit that activation overall free energy was reduced [38]. This behavior of thermodynamic is resembled to that which occurs when changing conformation of polymers, just like in denaturation of protein. It is totally differed from the observed properties of pectin acid hydrolysis [39]. Figured out that pectin's acid hydrolysis rate was inefficient to be the reason for non-enzymatic softening the tissue of the cucumber [40]. Identified salt, temperature, and calcium concentrations combined effects on fermented cucumber tissue's softening rate. The softening kinetics of fermented cucumbers did not follow the first-order simple reaction. Just like the tissues of many other plants, cucumber possesses enzymes that have the ability to degrade the ingredients of plant cell walls, which may lead to changing in the texture. In cucumbers, many activities of enzymes have been found such as exopolygalacturonase, pectinesterase, and endopolygalacturonase [41]. When fermenting or acidifying of cucumber, methyl groups are removed from pectin by pectinesterase [42]. Nevertheless, pectin's' enzymatic hydrolysis by polygalacturonases from cucumber has not been identified if it is a significant factor in fermented cucumbers' softening. Adding of fungal polygalacturonases into the tanks of fermentation, especially on the flowers attached to small cucumbers has been linked to the commercially importance of fermented cucumbers' enzymatic softening. Buescher and Burgin [43] developed a sensitive new method of diffusion plate to determine the activity of polygalacturonase in the brines of fermentation and found that alumino-silicate clay has the ability of adsorbing and removing the activity of polygalacturonase from the brines of fermentation that are recycled. Enzymes which could hydrolyze polysaccharides 31 Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 activities were found in olives, pears, and Semillon grapes. of the cucumbers cell wall have not studied widely comparing with the enzymes that degrade pectin [45]. Showed that the activity of endo-β-1,4- gluconase in cucumber is inhibited under pH of 4.8 while endoglucomannan-splitting enzyme retains its activity under pH of 4.0 but is inhibited within the fermentation. In fresh cucumbers, they characterized 6 enzymes which hydrolyze p-nitrophenylglycosides of β-dglucose, β-d-galactose, α-d-galactose, β-d-xylose, α-d-mannose, and α-l-arabinose, which were inhibited throughout the fermentation. The enzymes that have the ability to hydrolyze the synthetic substrates are widespread in plants. Resemble enzymatic Maruvada [44] and Takayanagi et al. [45] discovered the same p-nitrophenyl glycosidases detected by [44] in cucumbers. She reported undetectable levels in 2% NaCl brines throughout the first week of fermentation [46, 47]. Gathered calcium addition, fresh cucumbers' blanching relatively to enzyme inactivation, and a quick fermentation using a malolactic-negative Lactobacillus plantarum culture for cucumbers' fermentation and keeping a required texture in reduced (4%) sodium chloride concentration [48]. Found notable degradation products of glucosinolate in cucumbers fermented with Lactobacillus sakei compared to cucumbers manufactured with lactic acid bacteria starter cultures [49]. Reported that ascorbigen, a compound resulted from a degradation product reaction of indole glucosinolate (glucobrassicin) and ascorbic acid, is the cucumbers' dominant glucosinolate degradation product. Glucoraphinin existed in fresh cucumbers was converted over the fermentation into sulforphorane, however, sulforphorane was a relatively small glucosinolate degradation product in fermented cucumbers. There are many concerns about the biogenic amines' formation in cucumbers [50]. Reported that storing cucumbers up to 12 months led to the formation of tyramine. While very trace amounts of tryptamine, histamine, and spermine were determined. These findings were assured in a study on vegetable products which concluded that tyramine concentration was about 4.9 mg/100 g in canned cucumbers [51], and the same finding and the concentration reported by [50]. No health risk existed referring to these mentioned biogenic amine levels, with the possible exception that individuals taking medications possessing monoamine oxidase inhibitors. Compared to the fermentation of liquids such as beer, wine, and milk, unique problems are involved in the fermentation of whole vegetables. Structural integrity has to be preserved in whole vegetables, which is not a factor with liquids [52]. Tissue softening is also a serious defect that can be caused by pectinolytic enzymes of either microbial (primarily fungal) source [53] or of the cucumber fruit itself. Off-flavors and off-colors may result from improper methods of fermentation and handling. The cucumber pickle industry is faced with waste disposal, in addition to spoilage problems. These wastes consist of the salt used to prevent softening during fermentation and storage, and the organic wastes. Salt concentrations used greatly Thus, after storing the brine, the excess salt must be leached from the cucum- bers before they are processed into finished products. Disposal of this nonbiodegradable waste salt is a source of serious environmental concern. As the salt is extracted during leaching, soluble cucumbers, including desirable nutrients and flavor compounds, are also removed. These desirable components are not only lost, they must be degraded before being discharged into waterways. Discharge of salt and organic materials into municipal disposal systems typically entails an extra expense for pickle companies, since municipalities must charge for recovering the 1.6 Fermented cucumbers-related problems exceed the 2–3 percent desired in the final product [54]. cost of handling such waste [55] (Figure 3). #### Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Economic Values and Its Cultivation and Breeding understanding for softening of cucumber tissue. plant cell walls, which may lead to changing in the texture. of NaCl with various cations and anions on fermentation of sugar in cucumber juice. The most interesting thing, fructose was the most preferred fermentable sugar to Lactobacillus plantarum more than glucose in most of experiments. Along with addition of different salts, the utilization of sugar was decreasing as anion or cation concentrations increasing. Lu et al. [33] and Zhou et al. [34] have identified various volatile ingredients in cucumbers that fermented with Lactobacillus plantarum (2% NaCl). About 37 volatile ingredients were determined, and as a result of fermentation, there was a little change in most of them. Inhibition of (E, Z)-2,6 nonadienal and 2-nonenal production was the most outstanding fermentation effect on cucumber volatiles [35]. Characterized trans- and cis-4-hexenoic acid as the strongest odors that specify the brine aroma properties of commercially fermented cucumbers in nearly 6% NaCl [36]. Illustrated that exposing the slurries (2% NaCl) of fermented cucumber to oxygen resulting in formation of nonenzymatic hexanal plus a series of trans unsaturated aldehydes with 5–8 carbon atoms that linked with oxidized odor intensity development the tissue of fermented cucumber. In the existence of light, about 100 μg/ml concentration of calcium disodium EDTA preserve nonfermented pickles against bleaching of pigments and lipid oxidation [14]. Although, when using this compound, there was a little reduction in pickles' firmness retention. Firmness retention in cucumbers fermentation and storage is a key quality issue. It is difficult to assure the firmness retention (in reduced salt fermented cucumbers) equal to what can be accomplished by fermenting and storage in 6% NaCl or more. Nevertheless, over many previous years there was a wide Fleming et al. [21] showed the importance of calcium in keeping fermented cucumbers' firmness. It was found that first-order kinetics is followed by the nonenzymatic softening of acidified, blanched cucumber tissue [37]. The mentioned kinetic manner made it reasonable to identify the activation of entropy and enthalpy of cucumbers' nonenzymatic softening, although that the chemical reactions in charge of softening were not known. At 1.5 M NaCl, both activation of entropy and enthalpy were high. Cucumber softening was inhibited by calcium because it reduced activation entropy too much into a limit that activation overall free energy was reduced [38]. This behavior of thermodynamic is resembled to that which occurs when changing conformation of polymers, just like in denaturation of protein. It is totally differed from the observed properties of pectin acid hydrolysis [39]. Figured out that pectin's acid hydrolysis rate was inefficient to be the reason for non-enzymatic softening the tissue of the cucumber [40]. Identified salt, temperature, and calcium concentrations combined effects on fermented cucumber tissue's softening rate. The softening kinetics of fermented cucumbers did not follow the first-order simple reaction. Just like the tissues of many other plants, cucumber possesses enzymes that have the ability to degrade the ingredients of In cucumbers, many activities of enzymes have been found such as exopolygalacturonase, pectinesterase, and endopolygalacturonase [41]. When fermenting or acidifying of cucumber, methyl groups are removed from pectin by pectinesterase [42]. Nevertheless, pectin's' enzymatic hydrolysis by polygalacturonases from cucumber has not been identified if it is a significant factor in fermented cucumbers' softening. Adding of fungal polygalacturonases into the tanks of fermentation, especially on the flowers attached to small cucumbers has been linked to the commercially importance of fermented cucumbers' enzymatic softening. Buescher and Burgin [43] developed a sensitive new method of diffusion plate to determine the activity of polygalacturonase in the brines of fermentation and found that alumino-silicate clay has the ability of adsorbing and removing the activity of polygalacturonase from the brines of fermentation that are recycled. Enzymes which could hydrolyze polysaccharides 30 of the cucumbers cell wall have not studied widely comparing with the enzymes that degrade pectin [45]. Showed that the activity of endo-β-1,4- gluconase in cucumber is inhibited under pH of 4.8 while endoglucomannan-splitting enzyme retains its activity under pH of 4.0 but is inhibited within the fermentation. In fresh cucumbers, they characterized 6 enzymes which hydrolyze p-nitrophenylglycosides of β-dglucose, β-d-galactose, α-d-galactose, β-d-xylose, α-d-mannose, and α-l-arabinose, which were inhibited throughout the fermentation. The enzymes that have the ability to hydrolyze the synthetic substrates are widespread in plants. Resemble enzymatic activities were found in olives, pears, and Semillon grapes. Maruvada [44] and Takayanagi et al. [45] discovered the same p-nitrophenyl glycosidases detected by [44] in cucumbers. She reported undetectable levels in 2% NaCl brines throughout the first week of fermentation [46, 47]. Gathered calcium addition, fresh cucumbers' blanching relatively to enzyme inactivation, and a quick fermentation using a malolactic-negative Lactobacillus plantarum culture for cucumbers' fermentation and keeping a required texture in reduced (4%) sodium chloride concentration [48]. Found notable degradation products of glucosinolate in cucumbers fermented with Lactobacillus sakei compared to cucumbers manufactured with lactic acid bacteria starter cultures [49]. Reported that ascorbigen, a compound resulted from a degradation product reaction of indole glucosinolate (glucobrassicin) and ascorbic acid, is the cucumbers' dominant glucosinolate degradation product. Glucoraphinin existed in fresh cucumbers was converted over the fermentation into sulforphorane, however, sulforphorane was a relatively small glucosinolate degradation product in fermented cucumbers. There are many concerns about the biogenic amines' formation in cucumbers [50]. Reported that storing cucumbers up to 12 months led to the formation of tyramine. While very trace amounts of tryptamine, histamine, and spermine were determined. These findings were assured in a study on vegetable products which concluded that tyramine concentration was about 4.9 mg/100 g in canned cucumbers [51], and the same finding and the concentration reported by [50]. No health risk existed referring to these mentioned biogenic amine levels, with the possible exception that individuals taking medications possessing monoamine oxidase inhibitors.	doab	2025-04-07T03:56:59.115242	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 36 }
0071a6c6-eff7-449d-a548-4af9bff79a91.37	1.6 Fermented cucumbers-related problems Compared to the fermentation of liquids such as beer, wine, and milk, unique problems are involved in the fermentation of whole vegetables. Structural integrity has to be preserved in whole vegetables, which is not a factor with liquids [52]. Tissue softening is also a serious defect that can be caused by pectinolytic enzymes of either microbial (primarily fungal) source [53] or of the cucumber fruit itself. Off-flavors and off-colors may result from improper methods of fermentation and handling. The cucumber pickle industry is faced with waste disposal, in addition to spoilage problems. These wastes consist of the salt used to prevent softening during fermentation and storage, and the organic wastes. Salt concentrations used greatly exceed the 2–3 percent desired in the final product [54]. Thus, after storing the brine, the excess salt must be leached from the cucumbers before they are processed into finished products. Disposal of this nonbiodegradable waste salt is a source of serious environmental concern. As the salt is extracted during leaching, soluble cucumbers, including desirable nutrients and flavor compounds, are also removed. These desirable components are not only lost, they must be degraded before being discharged into waterways. Discharge of salt and organic materials into municipal disposal systems typically entails an extra expense for pickle companies, since municipalities must charge for recovering the cost of handling such waste [55] (Figure 3). Figure 3. Cucumber bloater defect caused by carbon dioxide microbiologically produced during fermentation by either yeasts or LAB [56].	doab	2025-04-07T03:56:59.115959	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 37 }
0071a6c6-eff7-449d-a548-4af9bff79a91.38	1.7 Preventing flavor formation Purge-and-trap analysis of cucumber slurries' volatile ingredients in 2 percent reduced-salt salt brine before and after cucumber fermentation. Volatile components' comparison before and after fermentation led to the derivation that the main influence of fermentation on volatile flavors was to prohibit the enzymatic production of E, Z-2,6-nonadienal and 2-nonenal enzymes in cucumbers [34]. These aldehydes are the major ingredients in charge of cucumbers' fresh flavor [57]. Although, after a few days of cucumber fermentation, when tearing the tissue of cucumber, the pH descends low enough to deactivate the enzymes that forming these compounds. In fresh cucumber slurries, just benzaldehyde, ethyl benzene, and o-xylene were not found within the volatile ingredients characterized in the fermented cucumbers. Recently, the absence of flavor influence of volatile aldehydes is the main effect of the fermentation on flavor [35]. In fermented pickled cucumber brines, a low influence of volatility flavor compound was characterized. Adding of saturated salt to brine samples and heating to 50 °C, SPME (solid-phase microextraction) fiber sampling followed by GC-olfactometry resulted in the identification of a component with an odor close to that of the fermentation brine. The component with a fermentation brine odor was characterized as trans-4-hexenoic acid. The existence of cis-4-hexenoic acid was also tentatively characterized. A solution containing 25 ppm trans-4-hexenoic acid, 10 ppm phenyl ethyl alcohol, 0.65 percent lactic acid, 0.05 percent acetic acid, and 8 percent sodium chloride in a reconstitute experiment had an odor very similar to that of fermented cucumber brine. Lactic acid, acetic acid, and sodium chloride concentrations are acceptable for commercial brines after completing the fermentation. Adding of phenyl ethyl alcohol resulted in in a few enhancements in the matching odor. For that, the key component in the simulated brine solution was trans-4-hexenoic acid. The source of trans-4-hexenoic acid in fermentation brines is, unfortunately, not recognized. 33 Author details Sarmad Ghazi Al-Shawi1 \* and Sadiq Jaafir Aziz Alneamah<sup>2</sup> 1 Food Science Department, Agriculture College, Basrah University, Basrah, Iraq 2 Food Science Department, Agriculture College, Kufa University, Al-Najaf, Iraq © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, \Address all correspondence to: [email protected] provided the original work is properly cited. Cucumber Pickles and Fermentations* DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Pickles and Fermentations DOI: http://dx.doi.org/10.5772/intechopen.96052 Cucumber Economic Values and Its Cultivation and Breeding 1.7 Preventing flavor formation Figure 3. yeasts or LAB [56]. Purge-and-trap analysis of cucumber slurries' volatile ingredients in 2 percent reduced-salt salt brine before and after cucumber fermentation. Volatile components' comparison before and after fermentation led to the derivation that the main influence of fermentation on volatile flavors was to prohibit the enzymatic production of E, Z-2,6-nonadienal and 2-nonenal enzymes in cucumbers [34]. These aldehydes are the major ingredients in charge of cucumbers' fresh flavor [57]. Although, after a few days of cucumber fermentation, when tearing the tissue of cucumber, the pH descends low enough to deactivate the enzymes that forming these compounds. In fresh cucumber slurries, just benzaldehyde, ethyl benzene, and o-xylene were not found within the volatile ingredients characterized in the fermented cucumbers. Recently, the absence of flavor influence of volatile aldehydes is the main effect of the fermentation on flavor [35]. In fermented pickled cucumber brines, a low influence of volatility flavor compound was characterized. Adding of saturated salt to brine samples and heating to 50 °C, SPME (solid-phase microextraction) fiber sampling followed by GC-olfactometry resulted in the identification of a component with an odor close to that of the fermentation brine. The component with a fermentation brine odor was characterized as trans-4-hexenoic acid. The existence of cis-4-hexenoic acid was also tentatively characterized. A solution containing 25 ppm trans-4-hexenoic acid, 10 ppm phenyl ethyl alcohol, 0.65 percent lactic acid, 0.05 percent acetic acid, and 8 percent sodium chloride in a reconstitute experiment had an odor very similar to that of fermented cucumber brine. Lactic acid, acetic acid, and sodium chloride concentrations are acceptable for commercial brines after completing the fermentation. Adding of phenyl ethyl alcohol resulted in in a few enhancements in the matching odor. For that, the key component in the simulated brine solution was trans-4-hexenoic acid. The source of trans-4-hexenoic Cucumber bloater defect caused by carbon dioxide microbiologically produced during fermentation by either acid in fermentation brines is, unfortunately, not recognized. 32	doab	2025-04-07T03:56:59.116009	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 38 }
0071a6c6-eff7-449d-a548-4af9bff79a91.39	Author details Sarmad Ghazi Al-Shawi1 \* and Sadiq Jaafir Aziz Alneamah<sup>2</sup> 1 Food Science Department, Agriculture College, Basrah University, Basrah, Iraq 2 Food Science Department, Agriculture College, Kufa University, Al-Najaf, Iraq \*Address all correspondence to: [email protected] © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.	doab	2025-04-07T03:56:59.116241	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 39 }
0071a6c6-eff7-449d-a548-4af9bff79a91.42	Cosmetic, Culinary and Therapeutic Uses of Cucumber (Cucumis sativus L.) Chidiebere Ugwu and Stephen Suru	doab	2025-04-07T03:56:59.116273	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 42 }
0071a6c6-eff7-449d-a548-4af9bff79a91.43	Abstract Cucumber (Cucumis sativus L.) is cultivated throughout the world as an important vegetable. This review offers an insight on the versatile use of cucumbers for cosmetic, culinary and therapeutic purposes. Epidemiological and nutritional studies have shown various benefits associated with the use of cucumber. As cosmetic, cucumber is popularly used for natural beautification and for skin treatments. As a vegetable, cucumber is the quintessential materials and indispensable for salads, soups and smoothie with diverse health benefits including weight loss, remedy for chronic constipation, anti-inflammatory, cardiovascular and cancerous diseases among others. The use including weight loss, cosmetic, culinary and therapeutic purposes. Keywords: cucumber, culinary, cosmetics, therapeutic, uses, health, benefits	doab	2025-04-07T03:56:59.116307	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 43 }
0071a6c6-eff7-449d-a548-4af9bff79a91.44	1. Introduction Cucumber (Cucumis sativus L.) is cultivated throughout the world because of its ability to thrive in both temperate and tropical conditions. As a result, fresh cucumbers are available throughout the year [1]. Historically, cucumber is one of the oldest cultivated crops and believed to be a native of the Asia continent [2]; parts of China with temperate climates and parts of southern regions of India with tropical climates. At present, cucumber is the fourth most widely cultivated vegetable crop in the world (after tomatoes, onions and cabbage) and China is by far the world's largest producer of it [1]. C. sativus belongs to the gourd family of Cucurbitaceous, which also includes cantaloupes, squash, pumpkins, melon and water melon [3, 4]. C. sativusis a tender creeping vine and hairy leaves with 3 to 5 pointed lobes. In general, it bears roughly cylindrical fruits and may be as large as 60 cm long and 10 cm in diameter with dark-green skin, crispy moisture rich flesh, and small edible seeds concentrated at its core [5]. There are close to 100 varieties, but common ones include the English, garden, Persian, mini, and lemon. Cucumbers are mainly eaten in the unripe green form when they taste sweet, have crunchy texture, and unique flavor. Thus, they are best-harvested young, tender and just short of reaching maturity. On getting to full maturity, the cucumber skin becomes tougher and turns yellow-white with an accompanied bitter and sour [1, 5]. Relative to other vegetables that have been widely researched and reported on, cucumber seems to have received little interest probably because of seemingly lack of concentrated levels of well-known bioactive compounds presents in garlic, onion, tomatoes and the likes. Generally, cucumbers are consumed because of their refreshing quality, in part due to their very high water content and crunchy texture. Studies have shown that cucumbers contain lignans, vitamin K, cucurbitacins and their derivatives (triterpenoids), flavonoids (apigenin, luteolin, quercetin, and kaempferol), antioxidants (beta carotene and vitamin C) and B vitamins among other trace elements and minerals [6–8]. With a 95% level of water content saturated with naturally-occurring nutrients and trace elements meshed in high dietary fiber, cucumbers are beginning to gain attention in therapeutic, culinary and cosmetic uses.	doab	2025-04-07T03:56:59.116390	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 44 }
0071a6c6-eff7-449d-a548-4af9bff79a91.45	2. Cosmetic uses of cucumber Cucumber has various benefits associated with skin treatments and natural beautification. As a cosmetic, cucumber is excellent for rubbing over the skin to keep it soft and white [9]. Because of its nutritious and an extremely cooling property, cucumbers are used by women to bringing cooling relief to the eyes in summertime [10]. Cucumber slices offer many topical benefits to the eyes and surrounding tissues through their hydrating properties, which work to reduce dehydration and their high levels of vitamin K that help reduce cutaneous eruption such as puffiness (eye bags) and dark shadows [9]. Cucumbers contain lignans that help to soothe down irritation and inflammation associated with sun burns and insect bites [11]. Cucumber fruit extracts are often incorporated as a primary ingredient in many topical skin preparations. Such preparations have been used as a moisturizer and skin toner by inhibiting tyrosinase [12]. Additionally, such preparations have been used to treat wrinkles and cleanse the skin. The deep cleansing action of cucumbers emanates from its naturally occurring organic acids such as glycolic, lactic and salicylic acids [9]. Glycolic and lactic acids are alpha hydroxyl acids used as chemical exfoliants that promote the natural removal of dead cells and to keep the protective surface layers healthy by dissolving the glue-like substance in the epidermal layer. The glue-like substance causes a buildup of dead skin cell layer; leaving skin dehydrated, dull and coarse. Histologically, alpha hydroxy acids have been shown to increase the thickness of the epidermis as well as cause increased collagen density, improved elastic fiber quality, increased papillary dermal thickness and increased dermal acid mucopolysacharide translating into thicker, healthier skin with fewer rhytids [13–15]. Alpha hydroxyl acids have been effective in the treatment of many skin conditions such as ache, psoriasis, bumps, pustules, eczema, dry skin, age spots, seborrheic keratosis, precancerous growths, hyperkeratosis, actinic keratosis and also black heads and whiteheads [16]. Glycolic acid is the most active and beneficial of the alphahydroxyl acids in skin care, because of its ability to penetrate through the cell wall by virtue of its small molecular size [17]. Once inside the cell, it triggers new formation of collagen and turns on the synthesis of dermal glycosaminoglycans to plump up the cell and the ground substance in the skin to reduce wrinkles on the skin's surface [18]. Lactic acid improves the appearance of photodamage and surface pigmentation [19]. Unlike glycolic and lactic acids, salicylic acid is a beta hydroxyl acid that exhibits a keratolytic, antiseptic and fungicidal properties [20]. It can be used for the treatment of hyperkeratotic and scaling conditions such as dandruff, ichthyosis and 41 Cosmetic, Culinary and Therapeutic Uses of Cucumber (Cucumis sativus L.) psoriaisis [21]. The fungicidal properties of salicylic acid may partly explain the topical use of cucumber preparation in the treatment of fungal skin infections such Cucumber soap is used by many women, and a cucumber wash applied to the skin after exposure to keen winds is extremely beneficial. It is used in preparation of glycerin and cucumber cream. Cucumber has use in perfume production [9]. Overall, cucumber and its preparations have become part of daily beauty product into face packs, facials, juice and many other things which can affect your skin [1]. Cucumber is the quintessential fruit that can be added to a variety of dishes. Typically, they are indispensable for salads or used for pickling, soups and smoothie especially in warm season or summer. Cucumber wedges tossed in a garden salad (consumed with fried and barbequed foods), slices on a sandwich, or used as an appetizer for parties have become the mainstay of many of today's lunches [1]. There are basically three separate uses for cucumbers: fresh whole, fresh sliced, and pickled. Regardless of variety, fresh whole cucumbers are grown for consumer retail sales. On the other hand, fresh sliced cucumbers are typically garden variety and are grown for the foodservice sector, which requires uniform sized slices for Pickling cucumbers tend to be smaller and thicker. The best known variety is the bumpy-skinned gherkin. Not all pickled forms of cucumbers are fermented. Fermented pickled cucumbers are made by combining cucumbers with water, salt, and bacteria and giving the bacteria the right amount of time to convert various substances in the cucumbers into different bioactive compounds that can elicit health benefits especially in the gastrointestinal tract. However, the unfermented pickled cucumbers, though labeled as pickles and usually sold in grocery, are made The consumption of fresh whole cucumbers has become a common trend among middle-class households in some African cultures. The fresh whole cucumbers are eaten alone as a snack or in conjunction with peanuts or peanut butter as an appe- In recent times, the cucumber diet have become increasingly popular and included among many sought-after choices because of its availability throughout the season, low calorific value and high dietary fiber as well as stress-free procedure associated with the conventional short-term weight reduction therapies [1]. In principle, there are no standard rules to this diet other than replacing most foods with cucumbers, along with a few protein-rich foods, such as eggs, chicken, lean meat, fish, cottage cheese and nuts. Since the diet lacks variety, it is considered extremely restrictive and unsustainable for a long-term with an attendant health risk. Traditionally, cucumber has been used in folk medicine to treat diseases such as diarrhea, diabetes, and hypertension. Consisting mostly water in which numerous electrolytes and phytochemicals are saturated, the unique chemical profile of cucumbers is thought to elicit a number of possible health benefits Notwithstanding, that some of these claims are still undergoing investigation, there is proven evidence that the phytoconstituents in cucumber possess chemopreventive and anticancer properties, antioxidant and anti-inflammatory properties [23–26]. by submerging cucumbers in a very acidic liquid (usually vinegar). tizer during folkloric/traditional display of hospitality. 4. Therapeutic uses of cucumber DOI: http://dx.doi.org/10.5772/intechopen.96051 3. Culinary uses of cucumber packaged salads and restaurant chain salad bars. as tinea [22]. Cosmetic, Culinary and Therapeutic Uses of Cucumber (Cucumis sativus L.) DOI: http://dx.doi.org/10.5772/intechopen.96051 psoriaisis [21]. The fungicidal properties of salicylic acid may partly explain the topical use of cucumber preparation in the treatment of fungal skin infections such as tinea [22]. Cucumber soap is used by many women, and a cucumber wash applied to the skin after exposure to keen winds is extremely beneficial. It is used in preparation of glycerin and cucumber cream. Cucumber has use in perfume production [9]. Overall, cucumber and its preparations have become part of daily beauty product into face packs, facials, juice and many other things which can affect your skin [1].	doab	2025-04-07T03:56:59.116681	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 45 }
0071a6c6-eff7-449d-a548-4af9bff79a91.46	3. Culinary uses of cucumber Cucumber Economic Values and Its Cultivation and Breeding cosmetic uses. bites [11]. salicylic acids [9]. also black heads and whiteheads [16]. 2. Cosmetic uses of cucumber Relative to other vegetables that have been widely researched and reported on, cucumber seems to have received little interest probably because of seemingly lack of concentrated levels of well-known bioactive compounds presents in garlic, onion, tomatoes and the likes. Generally, cucumbers are consumed because of their refreshing quality, in part due to their very high water content and crunchy texture. Studies have shown that cucumbers contain lignans, vitamin K, cucurbitacins and their derivatives (triterpenoids), flavonoids (apigenin, luteolin, quercetin, and kaempferol), antioxidants (beta carotene and vitamin C) and B vitamins among other trace elements and minerals [6–8]. With a 95% level of water content saturated with naturally-occurring nutrients and trace elements meshed in high dietary fiber, cucumbers are beginning to gain attention in therapeutic, culinary and Cucumber has various benefits associated with skin treatments and natural beautification. As a cosmetic, cucumber is excellent for rubbing over the skin to keep it soft and white [9]. Because of its nutritious and an extremely cooling property, cucumbers are used by women to bringing cooling relief to the eyes in summertime [10]. Cucumber slices offer many topical benefits to the eyes and surrounding tissues through their hydrating properties, which work to reduce dehydration and their high levels of vitamin K that help reduce cutaneous eruption such as puffiness (eye bags) and dark shadows [9]. Cucumbers contain lignans that help to soothe down irritation and inflammation associated with sun burns and insect Cucumber fruit extracts are often incorporated as a primary ingredient in many topical skin preparations. Such preparations have been used as a moisturizer and skin toner by inhibiting tyrosinase [12]. Additionally, such preparations have been used to treat wrinkles and cleanse the skin. The deep cleansing action of cucumbers emanates from its naturally occurring organic acids such as glycolic, lactic and Glycolic and lactic acids are alpha hydroxyl acids used as chemical exfoliants that promote the natural removal of dead cells and to keep the protective surface layers healthy by dissolving the glue-like substance in the epidermal layer. The glue-like substance causes a buildup of dead skin cell layer; leaving skin dehydrated, dull and coarse. Histologically, alpha hydroxy acids have been shown to increase the thickness of the epidermis as well as cause increased collagen density, improved elastic fiber quality, increased papillary dermal thickness and increased dermal acid mucopolysacharide translating into thicker, healthier skin with fewer rhytids [13–15]. Alpha hydroxyl acids have been effective in the treatment of many skin conditions such as ache, psoriasis, bumps, pustules, eczema, dry skin, age spots, seborrheic keratosis, precancerous growths, hyperkeratosis, actinic keratosis and Glycolic acid is the most active and beneficial of the alphahydroxyl acids in skin care, because of its ability to penetrate through the cell wall by virtue of its small molecular size [17]. Once inside the cell, it triggers new formation of collagen and turns on the synthesis of dermal glycosaminoglycans to plump up the cell and the ground substance in the skin to reduce wrinkles on the skin's surface [18]. Lactic acid improves the appearance of photodamage and surface pigmentation [19]. Unlike glycolic and lactic acids, salicylic acid is a beta hydroxyl acid that exhibits a keratolytic, antiseptic and fungicidal properties [20]. It can be used for the treatment of hyperkeratotic and scaling conditions such as dandruff, ichthyosis and 40 Cucumber is the quintessential fruit that can be added to a variety of dishes. Typically, they are indispensable for salads or used for pickling, soups and smoothie especially in warm season or summer. Cucumber wedges tossed in a garden salad (consumed with fried and barbequed foods), slices on a sandwich, or used as an appetizer for parties have become the mainstay of many of today's lunches [1]. There are basically three separate uses for cucumbers: fresh whole, fresh sliced, and pickled. Regardless of variety, fresh whole cucumbers are grown for consumer retail sales. On the other hand, fresh sliced cucumbers are typically garden variety and are grown for the foodservice sector, which requires uniform sized slices for packaged salads and restaurant chain salad bars. Pickling cucumbers tend to be smaller and thicker. The best known variety is the bumpy-skinned gherkin. Not all pickled forms of cucumbers are fermented. Fermented pickled cucumbers are made by combining cucumbers with water, salt, and bacteria and giving the bacteria the right amount of time to convert various substances in the cucumbers into different bioactive compounds that can elicit health benefits especially in the gastrointestinal tract. However, the unfermented pickled cucumbers, though labeled as pickles and usually sold in grocery, are made by submerging cucumbers in a very acidic liquid (usually vinegar). The consumption of fresh whole cucumbers has become a common trend among middle-class households in some African cultures. The fresh whole cucumbers are eaten alone as a snack or in conjunction with peanuts or peanut butter as an appetizer during folkloric/traditional display of hospitality. In recent times, the cucumber diet have become increasingly popular and included among many sought-after choices because of its availability throughout the season, low calorific value and high dietary fiber as well as stress-free procedure associated with the conventional short-term weight reduction therapies [1]. In principle, there are no standard rules to this diet other than replacing most foods with cucumbers, along with a few protein-rich foods, such as eggs, chicken, lean meat, fish, cottage cheese and nuts. Since the diet lacks variety, it is considered extremely restrictive and unsustainable for a long-term with an attendant health risk. #### 4. Therapeutic uses of cucumber Traditionally, cucumber has been used in folk medicine to treat diseases such as diarrhea, diabetes, and hypertension. Consisting mostly water in which numerous electrolytes and phytochemicals are saturated, the unique chemical profile of cucumbers is thought to elicit a number of possible health benefits Notwithstanding, that some of these claims are still undergoing investigation, there is proven evidence that the phytoconstituents in cucumber possess chemopreventive and anticancer properties, antioxidant and anti-inflammatory properties [23–26]. Cucumber has also been reported to modify plasma lipid and act as an analgesic [27]. Cucumbers are good sources of more than 73 different phenolic compounds known to elicit health benefits.	doab	2025-04-07T03:56:59.117176	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 46 }
0071a6c6-eff7-449d-a548-4af9bff79a91.47	4.1 Hydration On a daily basis, adequate hydration is crucial for healthy living and prevention of diseases especially the likes of constipation and kidney stones. Cucumber is a good source of superior hydration due to high content of water (95%) saturated with naturally-occurring electrolytes. Consumption of cucumbers offers the cells the much-needed hydration and vital nutrients required for optimal cellular functioning, repair and maintenance of membrane integrity [28, 29]. Thus cucumbers can help prevent dehydration during summer time or during and after exercises. Cucumber extract when pharmacologically refined has been reported to have the potential in the treatment of corneal acid burn through its hydrating properties [9].	doab	2025-04-07T03:56:59.117286	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 47 }
0071a6c6-eff7-449d-a548-4af9bff79a91.48	4.2 Body weight management Cucumbers have high content of dietary fiber and very low calorific value resulting from low carbohydrates and very low amount of protein and fat contents. Consumption of cucumbers can help to heighten satiety and naturally curb appetite, which make it easier to cut down on food intake. Cucumbers also improve digestion because of their high fiber content, and adequate digestion has been linked with easier weight loss. In view of the aforementioned, the cucumber diet has emerged and has been included among many sought-after choices in short-term weight loss therapeutic regimens [1]. However, it should be noted that cucumber diet are usually restrictive in variety and as such must be complemented with some protein-rich foods.	doab	2025-04-07T03:56:59.117360	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 48 }
0071a6c6-eff7-449d-a548-4af9bff79a91.49	4.3 Bone health Cucumber contains calcium, phosphorus and vitamin K. Vitamin K helps to improve calcium absorption. A sufficient intake of these elements has been associated with maintenance of healthy bones that are less likely to fracture especially among the elderly. Put together, these nutrients contribute to good bone health.	doab	2025-04-07T03:56:59.117430	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 49 }
0071a6c6-eff7-449d-a548-4af9bff79a91.50	4.4 Management of blood glucose level Cucumbers have a low score on glycemic index (GI), indicating that they provide important nutrients without or with minimal carbohydrates that can cause a spike in blood glucose level [30]. Besides, a recent report suggested that cucurbitans in cucumber can stimulate the release of insulin and regulate the metabolism of a key hormone in the processing of blood glucose and hepatic glycogen [31]. Put together, this may suggest that cucumbers may help in the control and prevention of diabetes, given credence to folkloric claim.	doab	2025-04-07T03:56:59.117473	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 50 }
0071a6c6-eff7-449d-a548-4af9bff79a91.51	4.5 Potential anti-cancer health benefit Cucumbers contain high levels of triterpen family of phytochemicals known as cucurbitacins. The consumption of cucumber avails us cucurbitacins A, B, C, D, and E, which may help prevent cancer by stopping cancer cells from proliferating and surviving. Cancer research studies have proved that the JAK–STAT and MAPK signaling pathways involved with cancer cell development and survival can be 43 Cosmetic, Culinary and Therapeutic Uses of Cucumber (Cucumis sativus L.) cancer risk of the ovary, prostate, breast and uterus [34]. 4.6 Cardiovascular health benefits of cucumber low density lipoproteins among others [26]. 4.8 Other health benefits of cucumber 4.7 Antioxidant and anti-inflammatory benefits of cucumber of- MDA and 4-HNE [39]. blocked through the effects of cucurbitacins [24, 25]. While there are currently no current anti-cancer therapies that utilize cucurbitacins, experimental research has The results of cancer studies have shown that cucumber lignans such as lariciresinol, pinoresinol and secoisolariciresinol are converted by intestinal bacteria into enterolignans. These enterolignans including enterodiol and enterolactone have been reported to bind to estrogen receptors thereby eliciting both pro-estrogenic and anti-estrogenic effects [33]. Some preliminary results have shown that consumption of plant derived lignans including cucumbers can reduce estrogen-related There are several processes through which cucumber consumption may elicit cardiovascular benefits. Cucumbers are good sources of dietary fiber, particularly in their skins. Dietary fibers are known to significantly reduce the absorption of dietary cholesterol thus positively modifying the blood lipid profile with an attendant reduction in cholesterol buildup in the arteries. More so, cucumbers provide potassium and magnesium that may contribute to preventing high blood pressure [9, 35, 36]. With 73 different phenolic constituents, cucumber provides protection against oxidative insults for the blood vessels and their vulnerable contents such as The cucurbitacins in cucumber may also help prevent atherosclerosis. There have been reports on Cucurbitacin B and E in glycosidic form to exhibit inhibitory effect on lipid oxidation products like malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [37, 38]. These positive reports bolster the therapeutic role of cucurbitacins in artherosclerosis, which involves modification of lipoproteins by involvement A good number of phytochemicals present in cucumber have been reported to exhibit antioxidant and/or anti-inflammatory activities via sparing effect on other antioxidants and/or regulation of antioxidant enzymes in metabolic pathways involved. Small-scale human studies have been conducted using some of these identified phytochemicals in cucumber and found to provide some health benefits [29]. The cyclooxygenase 2 (COX 2), a pro-inflammatory enzyme, has been shown to be inhibited by cucumber extract [34]. In addition, antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase showed increased activities in studied participants that consumed cucumber powder. Some studies have shown that cucumber can reduce the generation of reactive oxygen species (ROS) and reactive carbonyl species (RCS), which could be of help in individuals with type 2 diabetes. The high content of phenolic compounds (flavonoids and terpenoids) present in cucumbers may be involved in this probable health benefit by significantly lowering the levels of ROS and RCS. Its high content of fisetin flavonoid gives it the potential anticancer, antioxidant and anti-inflammatory benefits [40]. Since cucumbers are rich in phytonutrients, there could be logical theoretical link between their consumption and amelioration of some chronic diseases. The peel and seeds are the nutrient dense parts of cucumber and contain beta-carotene that is good for the eyes [41]. Cucumber provides an alkaline diet, yielded promising results awaiting confirmation in human studies [31, 32]. DOI: http://dx.doi.org/10.5772/intechopen.96051 #### Cosmetic, Culinary and Therapeutic Uses of Cucumber (Cucumis sativus L.) DOI: http://dx.doi.org/10.5772/intechopen.96051 blocked through the effects of cucurbitacins [24, 25]. While there are currently no current anti-cancer therapies that utilize cucurbitacins, experimental research has yielded promising results awaiting confirmation in human studies [31, 32]. The results of cancer studies have shown that cucumber lignans such as lariciresinol, pinoresinol and secoisolariciresinol are converted by intestinal bacteria into enterolignans. These enterolignans including enterodiol and enterolactone have been reported to bind to estrogen receptors thereby eliciting both pro-estrogenic and anti-estrogenic effects [33]. Some preliminary results have shown that consumption of plant derived lignans including cucumbers can reduce estrogen-related cancer risk of the ovary, prostate, breast and uterus [34].	doab	2025-04-07T03:56:59.117533	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 51 }
0071a6c6-eff7-449d-a548-4af9bff79a91.52	4.6 Cardiovascular health benefits of cucumber Cucumber Economic Values and Its Cultivation and Breeding known to elicit health benefits. 4.2 Body weight management protein-rich foods. 4.4 Management of blood glucose level of diabetes, given credence to folkloric claim. 4.5 Potential anti-cancer health benefit 4.3 Bone health 4.1 Hydration Cucumber has also been reported to modify plasma lipid and act as an analgesic [27]. Cucumbers are good sources of more than 73 different phenolic compounds On a daily basis, adequate hydration is crucial for healthy living and prevention of diseases especially the likes of constipation and kidney stones. Cucumber is a good source of superior hydration due to high content of water (95%) saturated with naturally-occurring electrolytes. Consumption of cucumbers offers the cells the much-needed hydration and vital nutrients required for optimal cellular functioning, repair and maintenance of membrane integrity [28, 29]. Thus cucumbers can help prevent dehydration during summer time or during and after exercises. Cucumber extract when pharmacologically refined has been reported to have the potential in the treatment of corneal acid burn through its hydrating properties [9]. Cucumbers have high content of dietary fiber and very low calorific value resulting from low carbohydrates and very low amount of protein and fat contents. Consumption of cucumbers can help to heighten satiety and naturally curb appetite, which make it easier to cut down on food intake. Cucumbers also improve digestion because of their high fiber content, and adequate digestion has been linked with easier weight loss. In view of the aforementioned, the cucumber diet has emerged and has been included among many sought-after choices in short-term weight loss therapeutic regimens [1]. However, it should be noted that cucumber diet are usually restrictive in variety and as such must be complemented with some Cucumber contains calcium, phosphorus and vitamin K. Vitamin K helps to improve calcium absorption. A sufficient intake of these elements has been associated with maintenance of healthy bones that are less likely to fracture especially among the elderly. Put together, these nutrients contribute to good bone health. Cucumbers have a low score on glycemic index (GI), indicating that they provide important nutrients without or with minimal carbohydrates that can cause a spike in blood glucose level [30]. Besides, a recent report suggested that cucurbitans in cucumber can stimulate the release of insulin and regulate the metabolism of a key hormone in the processing of blood glucose and hepatic glycogen [31]. Put together, this may suggest that cucumbers may help in the control and prevention Cucumbers contain high levels of triterpen family of phytochemicals known as cucurbitacins. The consumption of cucumber avails us cucurbitacins A, B, C, D, and E, which may help prevent cancer by stopping cancer cells from proliferating and surviving. Cancer research studies have proved that the JAK–STAT and MAPK signaling pathways involved with cancer cell development and survival can be 42 There are several processes through which cucumber consumption may elicit cardiovascular benefits. Cucumbers are good sources of dietary fiber, particularly in their skins. Dietary fibers are known to significantly reduce the absorption of dietary cholesterol thus positively modifying the blood lipid profile with an attendant reduction in cholesterol buildup in the arteries. More so, cucumbers provide potassium and magnesium that may contribute to preventing high blood pressure [9, 35, 36]. With 73 different phenolic constituents, cucumber provides protection against oxidative insults for the blood vessels and their vulnerable contents such as low density lipoproteins among others [26]. The cucurbitacins in cucumber may also help prevent atherosclerosis. There have been reports on Cucurbitacin B and E in glycosidic form to exhibit inhibitory effect on lipid oxidation products like malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [37, 38]. These positive reports bolster the therapeutic role of cucurbitacins in artherosclerosis, which involves modification of lipoproteins by involvement of- MDA and 4-HNE [39]. ## 4.7 Antioxidant and anti-inflammatory benefits of cucumber A good number of phytochemicals present in cucumber have been reported to exhibit antioxidant and/or anti-inflammatory activities via sparing effect on other antioxidants and/or regulation of antioxidant enzymes in metabolic pathways involved. Small-scale human studies have been conducted using some of these identified phytochemicals in cucumber and found to provide some health benefits [29]. The cyclooxygenase 2 (COX 2), a pro-inflammatory enzyme, has been shown to be inhibited by cucumber extract [34]. In addition, antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase showed increased activities in studied participants that consumed cucumber powder. Some studies have shown that cucumber can reduce the generation of reactive oxygen species (ROS) and reactive carbonyl species (RCS), which could be of help in individuals with type 2 diabetes. The high content of phenolic compounds (flavonoids and terpenoids) present in cucumbers may be involved in this probable health benefit by significantly lowering the levels of ROS and RCS. Its high content of fisetin flavonoid gives it the potential anticancer, antioxidant and anti-inflammatory benefits [40]. Since cucumbers are rich in phytonutrients, there could be logical theoretical link between their consumption and amelioration of some chronic diseases. #### 4.8 Other health benefits of cucumber The peel and seeds are the nutrient dense parts of cucumber and contain beta-carotene that is good for the eyes [41]. Cucumber provides an alkaline diet, and due to its triterpene content, works well in regulating diseases of the immune system [42]. Majority of alkaline fruits and vegetables like cucumber are also antiinflammatory in nature [43] and thus neutralize the body's acid pH specifically in the kidneys [29].	doab	2025-04-07T03:56:59.117823	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 52 }
0071a6c6-eff7-449d-a548-4af9bff79a91.53	5. Conclusions The cosmetic and culinary uses of cucumber without any reported adverse effect are indicative of its versatility. Cucumbers are endowed with phytochemicals that have been reported to elicit positive health benefits ranging from hydration of body cells, body weight control and management of degenerative diseases. It is hoped that as more clinical researches are conducted on whole extracts and phytoconstituents, amazing health benefits shall be unfolded and folkloric claims shall be clarified for diverse but specific therapeutic uses.	doab	2025-04-07T03:56:59.118020	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 53 }
0071a6c6-eff7-449d-a548-4af9bff79a91.54	Conflict of interest The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.118080	1-12-2023 19:39	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0071a6c6-eff7-449d-a548-4af9bff79a91", "url": "https://mts.intechopen.com/storage/books/9704/authors_book/authors_book.pdf", "author": "", "title": "Cucumber Economic Values and Its Cultivation and Breeding", "publisher": "IntechOpen", "isbn": "9781839680243", "section_idx": 54 }

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